Micro 320, Spring 2016, Handout
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Class 1 COURSE INTRODUCTION
MICRO 320 Molecular and Cellular Bacteriology MWRF, 10-10:50 AM, 1420 Molecular
Biology Building
Contacting the instructor
Thomas A. Bobik, Professor BBMB
2152 Molecular Biology Building
bobik@iastate.edu
(515) 294-8247
Office hours: WRF 11 AM-12 noon.
Course web site for Prof Bobik's part only.
Blackboard.
Grading
First half of the semester with Dr. Bobik as instructor
250 points total
Two exams (125 pts each). Essay, short answer and multiple choice.
Friday. Feb. 5, normal class time and room
Wednesday, Mar. 2, normal class time and room
Letter grades will be 90% (A), 80% (B), 70% (C), 60% (D), and <60% (F) with
a curve possible based on student performance and fairness.
Exam content.
Most exams questions (~90%) will come from the course notes and you
should focus your studies on the notes. A few questions (~10%) will come from
material presented in class but not in the notes.
Micro 320, Spring 2016, Handout
Instructor's background
B.S., Microbiology, Indiana University, Bloomington, IN
M.S., Microbiology, University of Illinois, Urbana, IL
Ph.D., Microbiology, University of Illinois, Urbana, IL
Instructor for Microbial Diversity Course at Woods Hole, MA
Post-Doctoral Studies, Microbial Genetics, University of Utah, Salt Lake City
Assistant Professor of Microbiology at the University of Florida, Gainesville, FL
Instructor for Introductory Microbiology
Instructor for Molecular Genetics
Professor, ISU, BBMB Department
Instructure for MICRO 320 Molecular and Cellular Bacteriology.
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Instructor for MICRO 551, Microbial Diversity and Phylogeny.
Instructor for MICRO 430, Prokaryotic Diversity and Ecology.
Instructor for BBMB 404, Biochemistry.
Visiting professor, UCLA, Dept of Chemistry and Biochemistry: X-ray crystallography
Current research by Professor Bobik
1. Bacterial microcompartments.
2. Genetic engineering E. coli for the production of renewable chemicals.
Main course topics
1. The key molecular process required to build a new bacterial cell
2. Molecular repair, targeting and homeostasis systems
3. Genetic systems in bacteria
4. How bacteria respond to their environments
5. Modern methods in bacteriology including reductionist and systems
biology approaches.
The main topics of this course are critical in many areas of microbiology,
biotechnology and medicine. Hence, this is a required course and core component of
the microbiology program at ISU.
Course themes
1. All cells on Earth are related at the molecular and cellular level.
2. Everything in this course is a paradigm that will help you develop intuition
about how biology systems work at the cellular and molecular level.
Micro 320, Spring 2016, Handout
CLASS 2 CELL STRUCTURE, COMPOSITION AND REPLICATION
The Escherichia coli paradigm. This course will focus on Escherichia coli. It is the best
understood organisms on Earth and many of the principles that apply to E. coli apply to all
organisms. This is because all life on Earth evolved from a single cell or a small group of
coevolving cells and evolution proceeds by modification of existing systems. Hence,
everything in this course is a paradigm for understanding biology.
One of the goals of the course is to understand the key molecular processes needed to
replicate a bacterial cell, so let's take a look at this challenge in terms of the main
components of a bacterium the need to be replicated.
Many different types of bacteria are share basic cellular structures including
cell membranes, cell wall, flagella, pili, etc.
Transmission electron micrographs of E. coli (a gram negative bacterium)
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Most bacteria can be classified as either gram positive (G+) or gram negative (G-)
based on the composition of their cell envelopes.
E. coli is gram negative which is a major class of bacteria.
Summary of the G- cell envelope
Summary of the G+ cell envelope
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The chemical composition of Bacterial cells
All cells on Earth have a similar chemical composition. For E. coli and other cells to
replicate, they must obtain all the elements needed to build a cell from nutrients in its
environment. Some nutrients can be used directly to build new cells other nutrients must
be converted to biosynthetic building blocks via metabolism.
Elemental Compostition of E. coli (dry weight, E. coli is about 70% water)
50% Carbon
3% Phosphorous
20% Oxygen
2% Na, K, Sulfur
14% N
8% H
0.05% Ca, Mg, Cl
0.2% Fe
E. coli requires small, nonpolymeric organic molecules for growth and replication. A
typical organism will make a few thousand different small molecules.
Metabolomics. Attempts to measure all the small molecules present in a cell or a
subcellular structure at once. Most commonly, chromotography is used in conjunction with
mass spectrometry.
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E. coli and other cells use central metabolism to produce the small molecules
(precursors) required for growth from nutrients such as glucose and ammonia that are
present in its environment.
1. Nutrients are taken up from the environment by transport and diffusion.
2. Central metabolism is used to produce the energy and all the biosynthetic precursors
needed for growth. E. coli can be synthesized from 13 organic compounds that are
intermediates of central metabolism.
E. coli is a heterotroph (like us). It obtains carbon and energy for growth from organic
compounds like glucose.
This is different from autotrophs which obtain carbon from CO2 (which is an inorganic
compound dispite being made in part of carbon) and energy from sunlight or the oxidation
of inorganic compounds.
Micro 320, Spring 2016, Handout
To replicate, E. coli must be able to synthesize a variety of macromolecules.
The central dogma
DNA – RNA -protein
Transcription is the processes of producing RNA from a DNA template
Translation is the used to produce proteins from an mRNA template
Other macromolecules are synthesized by various biosynthetic pathways.
Under any given growth condition, E. coli will produce a few thousand proteins
PROTEOMICS seeks to measure all the proteins being produced by a cell under specified
conditions.
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2D gel electrophoresis of cytoplasmic proteins.
E. coli and all cells require a number of inorganic nutrients to grow. These are taken
up from the environment. Ions can have specific roles in catalysis by enzymes others fulfill
structural roles and roles in energy generation.
Example, carbonic anhydrase
requires Zn2+ for catalysis
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Bacterial growth
Bacteria grow logarithmically under ideal conditions and this process is quantified
using doubling times and specific growth rate constants.
Given the right nutrients, cells increase in cell size and mass, then replication of the
chromosome and cell division occur.
Most bacteria divide by binary fission.
Semi log plot of the number of bacteria versus time for a batch culture (semi-log
bacterial growth curve)
Quantifying bacterial growth
Doubling time-scientists quantify bacterial growth for many reasons, in industry when
bacteria are used to produce products fastest growth is often desired. Doubling time is
determined during the logarithmic phase of growth.
b = a x 2n
logb = loga + (nlog2)
n = (logb-loga)/log2
n= (logb-loga)/0.3010
b = total number of cells after time t
a = starting number of cells early in logarithmic growth
n = number of generations (doublings)
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Then the doubling time (g) = time/n
Specific growth rate constant and doubling time. µ is determined during log phase.
dN/dt = µN
ln N – ln No = µ(t-to)
divide both sides by 2.303
log10 N – log10 No = µ/(t-to)/2.303
N = the number of cells
µ = specific growth rate constant
µ = 0.693/g
µ has units of reciprocal time, for example h-1
Using semi log graph paper to determine µ and g
Slope of the exponential phase = (log10 N – log10 No)/(t-to) = µ/2.303
(Slope of the exponential phase) 2.303 = µ
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Gene and protein nomenclature is standardized in E. coli and other bacteria.
Usually, a 3 letter acronyms is used to name bacterial gene and proteins (applies to all
bacteria but not higher organisms). Genes in higher organisms have their own naming
systems.
For example, E. coli, the dnaA gene encodes the DnaA protein which acts as a positive
regulator of DNA replication. The polA gene encodes the PolA protein which is DNA
polymerase I.
By convention, genes are in italics, the first three letters are lower case and indicate
function, the fourth letter is a capital and indicated a specific gene. Proteins are normal
text with the first letter and the fourth letter (if present) capitalized.
Standard nomenclature is often used but not always in which case meaning must be
obtained from context.
The ecogene database provides a definition for all the acronyms for E. coli genes:
http://www.ecogene.org/gene/EG10746
CLASS 3 DNA REPLICATION
All living things use DNA as their hereditary material (The most common view
is that viruses are not alive).
To reproduce, E. coli must replicate its DNA with high fidelity. Errors in DNA
replication lead to mutations most of which are harmful.
A basic understanding of DNA structure helps explain DNA replication.
In dsDNA the single strands are antiparallel.
In dsDNA, bases interact by complementary base-pairing.
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Phosphodiester bond formation adds nucleotides to the growing DNA chain.
During DNA synthesis a phosphodiester bond is formed between the growing DNA a
strand and a nucleotide triphosphate substrate.
Bacterial genomes are supercoiled nucleoprotein complexes.
Prokaryotes typically have a single circular chromosome that is negatively supercoiled and
located in the nucleoid region.
Nucleoid. The central region of a prokaryotic cell where the DNA is found.
Supercoiling. The E. coli chromosome contains 30-200 supercoiled domains. The
supercoiling in these domains is independent of other domains.
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Histone-like, DNA-binding proteins in E. coli.
The E. coli chromosome s associated with a number of proteins including 4 small, basic,
histone-like proteins. Their likely function is to bend and compact DNA.
HUα
HUβ
HN-S
StpA
Prokaryotes do not have nucleosomes like those of eukaryotes.
REP elements.
Repetitive extragenic palindromes.
Palindromes are sequence that read the same in both directions and my form hairpin
structures by standard base-pairing.
About 21-65 bases in length
REPs may provide the binding sites needed for the formation of supercoiled domains.
REP elements also are thought to provide binding sites for DNA pol, DNA gyrase and IHF
and may fine tune transcription.
Micro 320, Spring 2016, Handout
DNA replication and function requires control of supercoiling.
Topoisomerases are used to control supercoiling in DNA
Type I cut one strand of DNA only, relaxation occurs (supercoils are lost) then the
strands are religated.
Type IA cleaves 1 strand of DNA passes it through the break and reseals.
Type IB cleaves 1 strand, mediates controlled unwinding then reseals
TypeII unwind or supercoil DNA by passing double stranded DNA through a doublestranded break.
DNA REPLICATION
DNA replication is essential for cell growth and reproduction
In nearly all bacteria, DNA replication starts at a single origin, proceeds bidirectionally
and ends at the terminus (theta replication).
DNA replication proceeds in the 5' to 3' direction (in other words the 3'end is
extended).
DNA replication is semiconservative.
DNA replication has a leading and a lagging strand
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DNA replication requires a variety of enzymes working in concert to unwind, stabilize,
prime and replicate DNA
The main enzymes involved in DNA replication in E. coli
Leading strand synthesis
Helicase
ssDNA binding protein
Primase
β-clamp/PolIII (β-clamp acts as a sliding clamp)
DNA gyrase
Lagging strand synthesis
Helicase
ssDNA binding protein
Primase
β-clamp/PolIII
RNAaseH (has 5’ to 3’ exonuclease activity the degrades the RNA primer)
PolI
ligase
DNA gyrase relaxes DNA ahead of the fork.
Diagram of one replication fork
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The replisome is a large multi-protein complex that carries out DNA replication in
living cells.
DnaG is primase, DnaB is helicase
The fidelity of DNA replication is primarily controlled by Watson-Crick base-pairing
and proof-reading by DNA polymerase.
Proofreading by PolIII
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DNA replication is carefully regulated to coordinate with cell division. In E. coli this
requires regulator proteins to selectively bind fully- and hemi-methylated DNA.
Initiation of DNA replication
Origin of replication-Initiation of DNA replication begins at the origin.
The origin is specific site on the bacterial chromosome, known as oriC, a 245 bp region
OriC contains several 13mer AT-rich sequences - which melt easily
OriC also contains five repeated DnaA binding boxes (a 9 bp sequence) where DnaA binds.
DnaA is a positive regulator of replication. DnaA forms a binary complex with ATP
(DnaA-ATP) which binds to five DnaA boxes found in OriC and helps to melt the A-T rich
repeats and facilitate the loading of helicase. DnaA preferentially binds to oriC when its
GATC sites are fully methylated.
Fully methylated DNA is DNA where both strands are methylated at GATC site.
When DNA PolIII becomes active it inhibits DnaA activity. Active DNA pol promotes
the hydrolysis of DnaA-ATP to DnaA-ADP which is inactive in initiating DNA replication
(the molecular details of why DnaA-ADP is inactive are not yet known).
The SeqA protein is a negative regulator of initiation. After the origin is replicated the
SeqA protein binds hemimethylated GATC sites (there are 11 in the origin) to inhibit
binding of DnaA and initiation. Over time the GATC sites are methyated by the Dam
methylase the competes with SeqA for these sites.
Hemimethylated DNA is made during DNA replications and exist for a short time before
becoming fully methylated again.
In addition, DnaA has lower affinity for hemimethylated DNA.
Biotechnology relevance
Some antibiotics specifically interfere with bacterial DNA replication by inhibiting DNA
gyrase.
An understanding of the principles of DNA replication underlies many biotechnology and
research applications.
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CLASS 4 TRANSCRIPTION
To grow and reproduce cells must be able to make many different RNA
molecules in the correct proportions.
Cells respond to environmental changes by changing their composition and
behavior. To do this, they must be able to control the amounts of the many
different RNA molecules present in the cell.
Cells make RNA by the process of transcription in which a DNA template is
copied to make a complementary RNA.
Transcription has three phases.
Initiation of RNA synthesis at a specific site
Elongation. Synthesis of RNA from a DNA template at 30-60 nucleotides/sec
Termination at a specific site
There are five important products of transcription in bacteria.
RNA polymerase is the enzyme that makes RNA from a DNA template. It consists of
multiple subunits and functional domains.
Holoenzyme
Core enzyme
Sigma factor
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The subunits of RNA pol are associated with particular functions.
α-subunit: assembly of the holoenzyme and recognition of transcriptional activators
β-subunit: catalytic sites of RNA polymerization
β’-subunit: DNA template binding
Sigma (σ)-subunit determines promoter specificity
Structure of RNA polymerase
Promoters are sites where RNA pol binds DNA to start transcription.
E. coli promoter. The typical E. coli sigma-70 promoter consensus promoter is a region
of DNA with a sequence similar to that shown below (sigma 70 is one of several alternative
sigma factors used by E. coli. The sequence does not need to be identical.
Consensus sequence of the E. coli sigma-70 promoter.
-35
-10
---TTGACA------------TATAAT---------AACTGT------------ATATTA-------
Micro 320, Spring 2016, Handout
RNA pol bound to a promoter. Note that the consensus promoter sequence is the
most common nucleotide at each position.
UP elements are used to increase the rate of transcription.
UP elements are a specific DNA sequence, generally centered at about base -42
Consensus UP element. −59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn −38
Increase transcription.
The sigma cycle
The sigma cycle is important to
gene regulation in E. coli.
Sigma factors are proteins that
determine the promoter specificity
of RNA pol.
Sigma factors cycle on and off of
RNA polymerase.
A sigma factor is required for
promoter binding by RNA
polymerase.
After 8-9 nucleotides are
transcribed sigma leaves RNA
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polymerase and is replaced by NusA.
When transcription terminates the core RNA polymerase and NusA are released.
Different E. coli sigma factors allow RNA pol to recognize different promoters.
Sigma
Consensus -35
Function
70
 (rpoD)
TTGACA
General housekeeping, most genes
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 (rpoH)
TNTCNCCTTGAA
Heat shock response
42/38

(rpoS)
CCGGCG
Stationary phase
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 (rpoN)
TTGGCACA
Nitrogen Assimilation
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 (rpoE)
GAACTT
Extreme heat shock; ECF,
Extracytoplasmic function responds to misfolded proteins or signals in the envelope
How are alternative sigma factors used to regulate transcription by bacteria?
Elongation occurs at 30-60 nucleotides per second
Termination of transcription can be
Rho-independent or Rho-dependent.
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Polarity.
Transcription factors (often called regulatory proteins in bacteria) are also used to
regulate transcription.
Supercoiling and transcription
Genomic view of transcription.
After transcription, RNA molecules are further processed, fulfill their cellular
function and are turned over.
Addition of PolyA tails to mRNA: A feature previously thought to be unique to
eukaryotic cells is the addition of poly(A) tails to the 3’ ends of many mRNA’s. Bacteria
also have poly(A) tails. May serve as a signal for mRNA degradation.
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tRNAs and rRNAs are often produced by processing of polycistronic transcripts into
final products. A polycistronic RNA corresponds to more than one gene.
Processing is required for the maturation of rRNA transcripts; these polycistronic
messages contain 16 S, 23S, and 5S rRNA and one or more tRNA’s interspersed in
intervening sequences/internally transcribed spacer.
Numerous RNases are involved, each recognizing specific sequences/regions of the
polycistronic message.
(Rnase P) is a Ribozyme (catalytic RNA), involved specifically in the removal of a 41 bp
fragment from the 5’-side of various tRNA precursor molecules
RNA decay:
tRNA (15-25%) and rRNA (70-80%) are stable. The secondary structure of rRNA and
tRNA protects them from degradation. Their half-lives are about 36 h.
mRNA (3-5%) is unstable. In contrast, mRNA half-life is approx. 40 sec at 37C. mRNA
stability affects protein production by translation.
When RNA is turned over it is broken down into nucleotides which are recycled. RNA
turnover is essential to gene regulation and cellular homeostasis.
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In laboratory research, transcription is often measured reporter genes.
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CLASS 5 TRANSLATION
Cells require several thousand different proteins to grow and reproduce. These are made
by translation of mRNA into protein
Bacterial mRNAs have 5 key
elements.
The genetic code is used to direct
proteins synthesis.
The genetic code is triplet and
non-overlapping and degenerate.
tRNA are used as adaptor molecules in translation.
Micro 320, Spring 2016, Handout
Amino acids must be activated for protein synthesis.
Amino acids are activated by attaching them
to tRNAs. The carboxyl group of the amino
acid is attached to the 3' hydroxyl group of
the terminal base at the 3, end of the
tRNA.
aminoacyl tRNA synthases are the enzymes
that add amino acids to tRNAs.
Add amino acid to tRNA
There is a distinct aminoacyl tRNA
synthase for each amino acid
Ribosomes are large nucleoprotein complexes used for protein synthesis.
Ribosome structure
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Translation has three stages
Initiation
Elongation
Termination
Initiation-p. 68
IF-1, IF-3, 30s form a complex
mRNA binds guided by the ShineDalgarno sequence and the AUG.
f-met-tRNA, IF-2-GTP join
The 50s binds and IF-3 leaves.
GTP is hydrolyzed in step 5 to eject
IF-1 and IF-2
f-met-tRNA (bacteria only)
met-tRNA (archaea and Eukarya)
Shine-Dalgarno sequence (a ribosome binding site (RBS) on mRNA.
The sequence of the RBS affects the rate of translation.
Elongation p. 69
16 amino acids per second at 37°C.
Role of elongation factor EF-T (EF-Tu/EF-Ts)
Ef-Tu brings aminoacyl-tRNAs to the A site then leaves
EF-T has two different subunits (EF-Tu/EF-Ts)
EF-Tu/EF-Ts + GTP → EF-Tu/GTP + EF-Ts
EF-Tu/GTP binds the next aminoacyl-tRNA to form a ternary complex
EF-Tu/GTP/aminoacyl-tRNA complex → A-site
GTP is hydrolyzed and EF-tu/GDP is ejected.
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One GTP is hydrolyzed per AA-tRNA bound
EF-ts then catalyzes a nucleotic exchange to reform EF-Tu/GTP (EF-Tu/GDP + EF-Ts +
GTP→ EF-Tu/GTP + EF-Ts + GDP)
Peptide bond formation
The 23s rRNA catalyzes peptidyl transfer
Amino acids are bound to tRNA by their carboxyl group.
The growing peptide is transferred to the amino group of
the amino acid carried by the incoming AA-tRNA. the
tRNA is the leaving group. The growth of the nacent
peptide is from the N-terminus to the C-terminus.
Translocation
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Termination of translation P. 72
UAA, UGA or UAG (stop codons) enter
the A-site.
Release factor RF-1 or RF-2 binds
inducing the peptidy transferase to
hydrolyze the peptide bound to tRNA in
the P-site and the peptide is released.
RF-3 removes RF-1 and RF-2.
Ribosome release factor (RRF) and EF-G
bind the A-site and mediates the
dissociation of the 70s ribosome into its
30s and 50s subunits while hydrolyzing
GTP.
Binding of IF-3 helps to mediate dissociation of the 30s subunit from the mRNA and
tRNA.
In some cases for polycistronic messages the mRNA does not dissociate but moves to the
next gene, a process known as translational coupling.
Selenocysteine
Proteins often require post-translational processing to become active and function.
Remove the formyl group of the N-terminal F-Met by Methionine deformylase or the
entire methionine.
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Translation requires a great deal of cellular energy. (As we will see later in the course,
bacteria produce proteins only when needed and only in the amounts needed to conserve
energy.)
Energy Expenditures in Translation
Most of the energy used in translation comes from GTP rather than ATP.





2 ATP equivalents are used by aminoacyl tRNA synthetase.
1 GTP is used for aminoacyl-tRNA binding.
1 GTP is used for translocation.
1 GTP is used to bind the large subunit to the small subunit initiation complex.
X GTP for ribosome dissociation
Polysomes
Translation of polycistronic mRNA
Transcription and translation are coupled in prokaryotes.
Polarity
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Growth rate-dependent control of ribosome synthesis p. 66
Microbes grow at different rates under different growth conditions. Faster growth
requires faster protein synthesis. Because the rate of peptide synthesis is constant,
faster protein synthesis requires more ribosomes per cell.
Transcription of rRNA operons requires a high NTP concentration. Excess ribosomes overconsume GTP and slow rRNA transcription. Lower levels of rRNA result in excess rproteins which in many casts repress their own translation.
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CLASS 6 PROTEIN FOLDING AND DEGRADATION
All cells have systems to aid protein folding and to degrade proteins into amino acids.
After proteins are released by the ribosome, they must fold into the proper 3-dimensional
structure in order to function. Some fold without assistance, others need the assistance
of chaperones. There are many incorrect folding pathways where a protein can become
trapped in a misfolded state.
Chaperones
Chaperones are proteins used to assist protein folding
Trigger factor. Trigger factor is
bound to ribosomes. It helps nascent
peptides fold properly by providing proline
isomerase. All prolines are made in the
trans configuration by the ribosomes, but
many are cis in the final protein. Because
isomerization is slow it can impede protein
folding and an isomerase is needed.
Besides proline isomerization, trigger
factor also helps proteins fold by other mechanisms but how it does so is unknown.
Isomerization of the tripeptide phospho-serine-proline-alanine. When this occurs in a
protein it turns the polypeptide.
DnaKJ GrpE
DnaK-ATP can assist protein
folding by binding to proteins to
and inhibiting nonproductive
folding pathways.
DnaK can also act as an
unfoldase. DnaK-ATP binds
hydrophobic portions of the
substrate loosely. DnaJ
stimulates the hydrolysis of ATP
to ADP. Then, DnaK-ADP binds
the substrate with high affinity
unfolding local regions.
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GrpE acts as a nucleotide exchange factor for DnaK, promoting the release of ADP and rebinding of ATP, along with the release of bound substrate.
GroEL GroES
The reaction cycle of the bacterial GroEL/GroES chaperonin system. The molecules are
shown in cross-sections. Each GroEL subunit consists of a central domain (red) an apical
substrate-binding domain (dark green) and a connecting intermediate region (yellow). The
binding of unfolded substrate protein (light blue) to the apical portion of GroEL is followed
by binding of ATP and the cochaperonin GroES (light green). The substrate is released into
the now-closed ring cavity where it can fold. ATP hydrolysis causes a conformational
change the makes the interior of the compartment more hydrophilic which promotes
protein folding. Binding of ATP to the opposite (trans) ring triggers the dissociation of
GroES and the dissociation of folded substrate protein from the complex. ATP hydrolysis
triggers binding-and release of the target protein. There are some indications that GroEL
may work as a “two piston” pump but exactly how it returns to the starting (ATP-free)
state is uncertain.
Molecular model of the GroEL chaperonin and the GroES co-chaperonin.
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GroEL/ES provide a protected environment in which proteins fold. ATP hydrolysis changes
the interior surface to more hydrophilic supported burial of hydrophobic residues in the
substrate protein.
Chaperonins are a subset of chaperones that have the stacked double ring structure like
GroEL
Chaperones are important for surviving stresses that cause protein unfolding such has
high temperature or extreme pH.
Chaperones and the heat shock response. Many chaperones are induced in response to
heat shock. Heat shock causes many proteins to unfold and the cell must eliminate
unfolded proteins to thrive.
DnaK (HSP70)
DnaJ (HSP40)
GroEL (HSP60
GroES (HSP10)
Protein Degradation
Protein degradation is essential to the health of all cells.
Misfolded proteins can be harmful to cells. Harsh conditions such as high temperature
or low pH can cause proteins to misfold.
Turnover of selected transcription factors is often needed to stop gene
induction/repression
Proteins without partners can also be harmful due to inappropriate interactions.
Degradation of normal proteins which have a half-life.
In higher organisms. protein turnover is also essential to immunity.
Proteins are ultimately broken down into amino acids which are reused.
ATP-dependent proteases such a Lon and Clp are responsible for most protein turnover in
E. coli. These proteases are composed of multimeric rings that form a central chamber and
an AAA+ ATPase that caps the chamber and acts as a gatekeeper. The ATPase recognizes
substrates unfolds them and feeds them into the central chamber where they are
degraded. Confining the protease within a chamber helps to protect normal proteins form
degradation.
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The Lon protease assembles into a hexameric ring structure. Each subunit has proteolytic
and ATPase domains. Lon is used to degrade many damaged proteins as well as some shortlived transcription factors (regulators).
The ClpP protease forms a 7-membered ring that houses a proteolytic core. ClpP can
associate with either the ClpX or ClpA ATPases to form the ClpAP and ClpXP proteases.
The protease is capped top and bottom with the six-membered ring ATPases, ClpX or ClpA
which are the gate keepers for translocation of proteins into the proteolytic chamber.
ClpAP degrades abnormal proteins and some transcription factors.
ClpXP is better at degrading specific proteins and works together with tmRNA to
disengage ribosomes stalled on damaged mRNA and to degrade truncated proteins
resulting from mRNA damage.
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tmRNA (encoded by the ssrA gene) is used to release ribosomes from damaged mRNA
Micro 320, Spring 2016, Handout
Proteolysis is often regulated and provides a mean by which bacteria respond to
changing environments.
Regulated proteolysis. In some cases, adaptor proteins are needed to bring certain
proteins to the ATPase subunit of an ATP-dependent protease. For example sigma-S is
delivered to ClpXP by the RssB protein.
Degradation of normal proteins is not a random process. In some cases the N-terminal
amino acids determines degradation rate.
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Summary of protein folding and degradation.
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CLASS 7 PROTEIN TRAFFICKING/LOCALIZATION.
For cells to function, particular proteins must be localized to particular places. This
includes the cytoplasm, the inner membrane, the outer membrane (gram-). In addition,
cells must be able to secrete proteins outside the cell.
The Sec pathway is a major pathway for protein secretion in E. coli. It is used to
insert proteins into the inner membrane or move them through the inner membrane into
the periplasm. The Sec pathway is also used in conjunction with other systems to excrete
a number of proteins into the environment or host cells. Proteins that span the membrane
are known as integral membrane proteins and many are involved in energy generation by
electron transport, uptake of nutrients or the pumping out of toxins such as antibiotics.
Many proteins are also found in the periplasm where the function in detoxification of
harmful substances, energy generation, and cell wall biosynthesis. Protein excreted into
the environment are often used to degrade polymeric nutrients (such as carbohydrates
and proteins) into monomers (sugars and amino acids) that can be transported into cells as
food (bacteria are unable to take up large polymers).
Signal sequences are used in the sec pathway. Many integral membrane proteins and
secreted proteins that have short signal sequences on their N-terminus that route them
to the proper destination. The Sec pathway targets these proteins
Signal recognition particle (SRP)
binds the signal sequences of
protein destined to be integral
cytoplasmic membrane proteins. SRP
is composed of at 54 kDa protein
(Fth) and a 4.5s RNA (Ffs). SRP
binds the signal sequence (10-30 Nterminal amino acids) of membrane
proteins just as their synthesis
begins and stalls the ribosome. The
SRP-ribosome complex binds FtsY
at the membrane and the membrane
protein is co-translationally
inserted into the membrane. This
can occur without SecYEG.
However, in some cases, the FtsYbound protein is delivered to
SecYEG for insertion in the
membrane. The signal sequence in
generally not cleaved.
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The Sec system can also work independently of the SRP system to target proteins to
the periplasm. Periplasmic proteins are synthesized and released into the cytoplasm
where they are recognized by the SecB chaperone. SecB binds to internal sequences of
the pre-secreted protein and not the signal sequence. SecB maintains pre-secreted
proteins in an unfolded state and pilots them to the inner membrane where they bind
SecA. SecA bound to a precursor protein inserts deeply into the SecYEG channel. ATP
hydrolysis then disinsterts SecA leaving 20-30 amino acids of the precursor in the
SecYEG pore. Following the initial ATP-dependent insertion the PMF provides the energy
for translocation of the remainder of the protein. Once the periplasm is reached, the
signal sequence is cleaved by signal peptidase (LepB).
Twin-arginine translocation is another important protein secretion system in E. coli.
Some proteins such as TorA (TMAO reductase) contain an N-terminal twin-arginine motif
(RRXFXK). Insertion of these proteins in the cytoplasmic membrane reqires TatABCE and
is independent of the Sec system. The TAT system is unique in that it can insert active
pre-folded proteins into the inner membrane. ATP hydrolysis is necessary for secretion.
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Twin Arginine Secretion System (TAT)
Insertion of proteins into the outer membrane of E. coli (Gram-). Evidence to date
indicates they are first localized to the periplasm by the Sec pathway and then find their
way to the outer membrane by an uncertain mechanism.
Secretion of proteins outside the cell into the environment or host cells. In many
cases, the Sec pathway is used to target proteins to the inner membrane and the
periplasm where they may remain or be further translocated to the outer membrane, the
surrounding environment, or host cells by other systems.
A variety of systems are used to secrete proteins outside of the cell into the
environment or into host cells.
Type I are similar to ABC transporters but has some additional components that span the
periplasm and out membrane. They bypass the Sec system and transport both small
molecules and proteins with proteins like being transported in the unfolded state.
Type II: sec-dependent-similar to pilus assembly. Proteins are pumped out with a pilus
used like a piston.
Type III: Similar to flagella assembly. The system acts like a syringe to inject proteins
into host cells.
Type IV: related to conjugation systems.
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TypeV are autotransporters
Type VI: related to phage
Type I secretion systems are similar to ABC transport systems but have some
additional components. The three major parts are an inner membrane gated channel
similar to ABC transport systems, and cytoplasmic protein that interacts with an outer
membrane channel, TolC. The inner membrane and cytoplasmic components are substrate
specific but TolC couples to many secretion systems for both proteins and small molecules.
The example shown below is for hemolysin excretion. Both ATP and the PMF are used an an
energy source. Type I secretion bypasses the Sec system. Proteins secreted by this
system often have an uncleaved C-terminal signal sequence.
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Type II secretion systems have many components in common with type IV pilus assembly.
Type IV pili are used in motility like grappling hooks. For secretion, the type IV-like
proteins work together with the Sec system. Sec delivers proteins to the periplasm where
the signal sequence is cleaved and the fold. Type II systems are thought to ram
presecredted protein through the outer membrane using a pilus like structure. Type II
systems are substrate specific but mechanism of specificity is uncertain.
Micro 320, Spring 2016, Handout
Type III secretion systems have many
similarities to flagella and are used to inject
proteins (including virulence factors) into host
cells. They are thought to work like a syringe.
Folded protein domains often have diameters
of 20 to 30 Å, suggesting that if effector
proteins were transported through the
interior of the needle, they would need to be
partially or fully unfolded, as discussed below.
Type III secreted proteins have N-terminal
(or 5’) signal sequences but it is uncertain
whether they are in the protein or RNA.
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Type IV secretion systems are related
to conjugations systems. Used by many
pathogens for delivering effector
proteins and virulence factors. Some type
IV sytems move proteins from the
cytoplasm outward, but others work in
conjuctin with the Sec system.
Type V secretion systems are autotransporters that mediate their own secretion across
the outer membrane after being localized to the periplasm by the Sec system, the Cterminal domains of these proteins bind the membrane and form a pore through which
protein is presumed to move then autoproteolysis of the C-terminus releases the protein
into the environment.
Type VI secretion systems are related to phage and are thought to punch holes in the
membrane using the same protein that phage such as T4 does, the VgrG protein.
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Two-partner secretion systems are a type of transporter in which for each protein to be
transported there is a specific channel-forming β-barrel transport protein. The are used
for transport across the outer membrane. Both proteins have a signal sequence and are
localized to the periplasm by the sec system. One protein inserts in the membrane using a
beta-barrel to form a channel. It specifically binds its partner and translocates it across
the outer membrane and into the environment. G- bacteria transport some virulence
factors in the way
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GENETICS DEFINITIONS AND TERMS
A lot of terminology is needed to provide a basis for disussing genetics.
Genotype: the precise sequence of nucleotides in the genome of an organism
Phenotype: the visible properties of an organism
Mutation: a heritable change in the base sequence of the DNA of an organism.
Mutant: a strain carrying a mutation.
Allele: A particular form of a gene. When a wild-type gene undergoes a mutation a new
allele is formed.
Wild-type: An organism, gene or protein, in its “normal” form which is used as the basis of
comparison in genetic research. Often an organism isolated from nature or the most
common allele of a gene found in nature or an allele designated by a scientist.
Point mutation: A mutation that changes a single base-pair in DNA.
Missense mutation: The change results in the insertion of a different amino acid into the
protein product
Nonsense mutation: The change results in the formation of a stop codon
Silent mutation: The change does not influence the protein sequence (the altered codon
encodes the same amino acid)
Deletions and insertions: the removal or addition of bases
Frameshift mutations: the deletion or insertion of one or more bases resulting in a shift
in reading frame
Polar mutations: a mutation that prevents expression of downstream genes that are in the
same operon as the original mutation.
Lethal mutations: mutations that result in the death of an organism under all growth
conditions.
Conditional lethal mutations: mutations in genes that are essential for growth under all
conditions, but that are lethal only under certain conditions.
Reversion mutation- a second mutation that restores the original or wild-type phenotype
to a mutant.
True reversion mutation = Back mutation - a change that restores the original nucleotide
sequence.
Suppressor mutation (second-site reversion)-a second mutation (at a different site from
the first mutation) that that restores the original or wild-type phenotype to a mutant..
Intragenic suppressor - a second mutation within the same gene that overcomes the
phenotype of the first mutation; e.g. a frame shift mutation that restores the original
frame
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Extragenic suppressor - a mutation in gene other than the one with the first mutation
that restores the original or wild-type phenotype
tRNA supressors, protein-protein binding supressors.
Revertant: Organism that has undergone a reversion mutation.
Prototroph: an organism that can grow on minimal medium.
Auxotroph: a mutant unable to grow on minimal medium but able to grow on rich medium or
on minimal medium supplemented with growth factors. Usually biosynthetic mutants.
Spontaneous mutations: mutations that arise naturally in cells due to normal chemical
processes that damage DNA resulting in base misincorporation during DNA replication.
Chemical process that damage DNA include the following”
Spontaneous conversion of C to U
Depurination to make apurinic sites.
DNA damage by reactive compounds formed from oxygen.
The frequency of spontaneous mutations is about 10-9-10-10/base pair/generation.
Induced mutations: mutations that result from exposure to a physical or chemical agent
called mutagens. Mutations are often induced for genetic research on a variety of
different systems.
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CLASS 8, GENETIC RECOMBINATION
Recombination is a process that generates new DNA molecules by genetic exchanges
that involve breaking are reforming phosphodiester bonds (the bonds that hold DNA
bases together).
The breaking and reforming of phosphodiester bonds in recombination is referred to
as genetic crossovers.
Recombination is essential to DNA repair and the generation of genetic diversity. It
is also an underlying basis of many genetic methods essential to scientific research.
Homologous recombination - genetic exchanges or rearrangements between DNA
sequences that are identical or nearly identical (i. e. homologous sequences) over longer
stretches, generally at least 50 bp although longer sequences are needed for a reasonable
frequency of recombination in many organisms.
Site-specific recombination aka illegitimate recombination- genetic rearrangements or
exchanges between sequence that have only short regions of identity (10-20 bp) or no
sequence identity.
Homologous recombination is a complex process - over 25 genes in E. coli are
required. The following are key components.
RecBCD- ATP-dependent helicase (unwinds DNA) + nuclease activity (degrades
DNA)
Chi- consensus octamer that occurs every 5-10 Kb- approximately 1,000 sites in E.
coli (its initial target for recombination). Its presence increases
recombination frequency and defines hotspots (E. coli Chi = 5’-GCTGGTGG-3’)
RecA- synaptase that facilitates alignment of homologous DNA sequences (also
functions outside of recombination as co-protease).
RuvAB protein promotes branch migration
RuvC protein resolves holiday junctions.
1.
RecBCD complex exhibits helicase (unwinds DNA) and nuclease activities. RecBCD
binds to the end of dsDNA and unwinds DNA. During unwinding it may also
degrade ssDNA although this is dependent on conditions in vitro and the in
vivo process is uncertain.
2.
When the sliding RecBCD complex contacts Chi site it introduces a nick on the
upper DNA strand (5’-3’), its nuclease acidity is inhibited and RecA synaptase
begins to load on the ssDNA to form a synaptic filament.
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3.
RecA now scans donor DNA for homology
4.
Once homology is found the single-stranded donor filament and double-stranded
recipient are aligned making a stable displacement loop is made in the
recipient strand. This leads to complementary base-pairing between
homologous regions.
5.
Resolution occurs through the formation of a Holliday junction- nucleases and
ligases facilitate making the recombinant molecules. There may be some
unpaired regions that will be repaired by the mismatch repair system
The activities of RecBCD are dependent on the conditions in vitro. This is why some
textbooks present different version of RecBCD function
Micro 320, Spring 2016, Handout
Model for homologous recombination.
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Micro 320, Spring 2016, Handout
Model for recombination from the textbook.
The RecA protein is particularly important for recombination. recA mutants are down
1000-fold for recombination. The remaining activity is from the RecF pathway.
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Homologous recombination is often reciprocal.
Some outcomes of recombination with single or double crossovers.
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CLASS 9 DNA REPAIR
Cells have a variety of DNA repair system.
DNA is often damaged during the normal life of cells and must be repaired to prevent
mutations most of which are harmful. Cells unable to repair DNA damage quickly die or are
transformed into cancer cells.
DNA damage results from a number of unavoidable processes such as exposure to free
radicals generated by oxidative metabolism, exposure to reactive chemicals and pollutants
and exposure to the sun or other UV light sources.
Proofreading by DNA polymerase
Photoreactivation repair is used to fix thymine dimers. P 152
Photolyase (phr) binds thymine dimers. Light in the 340-400 nm range must be absorbed
the Phr-dimer complex for photoreactivation to occur. Thymine dimmers are usually
formed by UV-irradiation of DNA.
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Nucleotide excision Repair (UvrABCD) p. 152
UvrAB scans DNA for structural distortions which result from mismatched bases or
thymine dimers. The DNA is bent, UvrA is ejected, UvrC is recruited and makes two single
stranded cuts, then the UvrD helicase displaces the single strand which is typically 12-13
bp in length. Pol I fills that gap and ligase seals the nick.
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Transcription-coupled repair.
The movement of RNA polymerase can be blocked by lesions in the template strand.
Transcription repair coupling factor (TRCF) binds to stalled RNA polymerase, displaces it
and recruits the excision repair enzymes, UvrABC.
Methyl-directed Mis-Match Repair (long-patch repair) (MutHLS, UvrD) p. 156
MutS binds to mismatched base pairs in DNA.
MutHL binds to a hemi-methylated GATC site looping out the DNA as needed
MutH cleaves the unmethylated strand near the GATC site.
UvrD unwinds the cleaved strand which is degraded by cellular nucleases that act on
ssDNA.
The gap is filled by PolI and sealed by DNA
ligase.
The Mut system is very good at repairing
transitions and frameshifts which are the
most common mutation caused during DNA
replication.
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Recombination Repair (RecA-dependent) (post-replication daughter strand gap repair)
p. 160
PolIII will sometime skip over damaged DNA and restart at the next priming site. This
leaves a single-stranded gap that can be repaired by recombination.
RecA mediates recombination that fills the gap with a single strand of DNA derived from
homologous DNA strand.
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Very short-patch mismatch repair (Me-cytosine repair). This is needed because MeCytosine is a hot spot for mutation, and many Me-Cyctosine exist in cells due to Dcm
methylation of CC(A/T)GG sites (not the same as Dam methylation which produces MeAdenine at GATC sites). MutS binds the mismatch. Vsr endonuclease cuts the DNA. MutL
recruits the UvrD helicase.
Base excision repair.
DNA glycosylases cleave the sugar-base bonds in DNA producing AP-sites (apurinic or
apyrimidinic sites). Specific endonucleases make a single-stranded cut near the AP-site
after which PolI fixes the damage using its 5’-3’ exonuclease and DNA polymerase
activities.
Types of lesions repaired by base excision repair (BER).
--Oxidized bases: 8-oxoguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG,
FapyA)
--Alkylated bases: 3-methyladenine, 7-methylguanine
--Deaminated bases: hypoxanthine formed from deamination of adenine. Xanthine formed
from deamination of guanine. (Thymidine products following deamination of 5methylcytosine are not repaired)
--Uracil inappropriately incorporated in DNA or formed by deamination of cytosine
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The mutator phenotype.
SOS “save our ship” response (UmuD’2C, LexA RecA)
The SOS response is induced by extensive DNA damage since this type of damage cannot
be repaired without causing lethal chromosome breaks. Under these conditions RecA,
which becomes activated by binding ssDNA, binds to LexA and induces autoproteolysis of
LexA. LexA is a repressor of many genes of the SOS response and autoproteolysis of
LexA results in their derepession. Proteins of the SOS response include UmuDC which are
the precursors of an error-prone DNA polymerase that can replicate DNA across lesions
caused by PolIII jumping over unrepaired DNA damage. RecA induces autoproteolytic
acitivity in UmuD leading to the formation of a truncated version ofUmuD (UmuD’). UmuD’
associates with UmuC to form UmuCD’2 which is a translesion error-prone DNA
polymerase. The SOS response increases mutagenesis because translesion DNA synthesis
involves the random incorporation of bases.
The SOS response also induces SulA which binds to FtsZ and blocks cell division which
results in the formation of long filamentous cells but allows more time for DNA repair.
After DNA damage is repaired LexA becomes stable accumulates and represses the SOS
response. Remaining SulA is degraded by the Lon protease.
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CLASS 9 RESTRICTION/MODIFICATION SYSTEMS AND TRANSPOSABLE
ELEMENTS
Restriction enzymes were discovered as enzymes that protect bacteria from invading
DNA by cutting it into pieces. They are best known for their importance in genetic
engineering.
Restriction endonucleases are enzymes that can cut dsDNA and internal sites as opposed
to exonucleases that degrade DNA starting from the ends. They usually recognize and cut
at a specific DNA sequence.
Bacteria use restriction enzymes to protect themselves from invading DNA that might be
harmful such as bacteriophage (bacterial viruses). An invading virus will be cut into pieces
and inactivated.
Host DNA is protected from self-restriction by methylation. Cells must protect the
genome from the own restriction enzymes. Usually the specific DNA sequence that the
restriction endonuclease recognizes is modified by the addition of a methyl group
(methylation) by a cognate methyltransferase.
Different bacteria (and strains of the same species) use different restriction
modification systems. Hence, DNA from bacterium A will be subject to degradation if it
enters bacterium B by some means.
There are 4 classes of restriction enzymes that vary in the subunit composition and
cofactor requirements.
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The methylases that modify host DNA to prevent self-restriction generally use
S-adenosylmethionine (SAM) as the methyl-group donor. SAM is a methyl donor in many
different cellular reactions.
Most restriction enzymes belong to class II.
Class II enzymes: (Example in diagram is EcoRI)
Separate endonuclease and methylase enzymes
Requires the involvement of SAM for the methylase
Recognition site requires dyad symmetry (two symmetrical parts), in the case of type
II restriction enzymes the recognition sequence reads the same on the
forward and reverse strands. - cleavage and modification at or near the
recognition site
Cleavage can result in blunt or cohesive (sticky) ends.
Micro 320, Spring 2016, Handout
In host cell the EcoRI methyltransferase methylated the EcoRI recognition sequence.
+ EcoRI + SAM →
The EcoRI system in composed of two genes found in an operon together, ecoRIR and
ecoRIM
TRANSPOSABLE GENETIC ELEMENTS
Transposable elements are DNA sequence that can move from place to place
(transpose) within a host genome. They widely distributed among bacteria, viruses,
and eukaryotic cells
Transposable elements are capable of transposition which is the process by which they
move from one place in a genome. Transposition is typically a rare event, occurring at
frequencies of once every 105 to 107 generations; thus, the genes of living organisms are
relatively stable.
A. Insertion sequences = IS elements are
the simplest form of transposable element.
They consist of a short sequence of DNA
flanked at each end by identical, or very
similar, sequences in reverse orientation,
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called inverted repeats about 15 to 25 bases long.
Between these repeats are genes that encode enzymes required for transposition, such as
the enzyme transposase, which recognizes the inverted repeats. Distinct insertion
sequences are called IS1, IS2, etc. (The number is in italics.)
B.
Transposons - transposable genetic elements that contain genes in addition to those
required for transposition, often genes for resistance to antibiotics. Transposons may
have a simple structure, in which short repeats flank the genes, or a complex, composite
structure in which two IS elements flank a central region containing the extra genes.
Transposons are typically labelled Tn#, such as Tn3 or Tn5. (The number is in italics.)
C. Bateriophage Mu is a lysogenic phage that uses transposition as one of its means of
replication.
Consequences of transposition: - The insertion of a transposon causes a mutation that will
likely inactivate the gene, promoter or terminator into which it is inserted.
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The two basic mechanisms of transposition are replicative and non-replicative.
Replicative transposition is characterized by the formation of a co-integrate in which the
two strands of the transposon become separated into ssDNA. The ssDNA is replicated by
host enzymes and homologous recombination separates the co-integrate.
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Nonreplicative transpostion involves excision of the transposon by a double-stranded
cleavage followed by integration into the target site.
Transposase is the enzyme that mediates transposition. A typical transposase cuts the
near the transposon end and at the target sites. Mediates synapse formation at the target
site (which may or may not be a specific DNA sequence) and ligates the transposon to the
target. Subsequent DNA repair used the host polymerase and ligase.
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CLASS 10 PLASMIDS AND DNA TRANSFER
Plasmids are relatively small, usually circular DNA molecules that exist independently
of host chromosomes. Thousands of different types of plasmids are known; over 300
different naturally occurring plasmids have been isolated from strains of E. coli alone.
Plasmids are probably very important to the ability of populations of microorganisms to
survive in the face of danger, such as in the presence of heavy metals or antibiotics
Some can transfer themselves; they are conjugative plasmids, i.e. they can transfer copies
of themselves to other bacteria during conjugation.
Plasmids often require partitioning systems to insure they are inherited during cell
division.
Plasmid partitioning is the distribution of plasmids to daughter cells following division. In
high-copy-number plasmids random segregation is sufficient to ensure the majority of
daughter cell contains plasmids. For low copy number plasmids active partitioning is needed
to assure that after replication each daughter cell gets a copy of the plasmid. Many
mechanisms are available, but two are particularly noteworthy.
Par cassettes
Plasmids that use that Par system replicate
at the middle of the cell.
A cis-acting telomere like site on the
plasmid (parS) contains ParR binding motifs.
A trans-acting protein ParR diffuses in the
cytosol and binds ParS
ParM is thought to form a filament that
pushes plasmids to opposite poles of the
cell using ATP to provide energy. ParM is a
homolog of actin a eukaryotic protein that
mediates pseudopod formation as well as
movement of some vesicles by
polymerization.
Addiction Modules- Toxin-antitoxin-loci
post segregation killing of plasmid-free
cells.
Toxins are stable (long lived) (e.g.,
mazF)
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Anti-toxins (antidote) are unstable (short-lived) (mazE)
Toxins are neutralized by forming a protein-protein complex with the anti-toxin
Targets include: Inhibition of DNA gyrase or initiation of replication at oriC
In general, two plasmids with the same origin of replication cannot exist in the same
cell at the same time.
Plasmid Incompatibility: - Two plasmids that cannot exist stably in the same cell are said
to be incompatible. In cases, that have been studied, incompatible plasmids have the same
mechanism of replication (same origin of replication) and/or the same system of
segregation. Random selection of plasmids from a pool of plasmids in a cell for replication
or segregation leads distortions in copy number then to pure lines.
Plasmid often confer traits that are conditionally important i.e important only under
certain conditions such as when a toxic compound is present.
Resistance plasmids are plasmids that carry gene which confer resistance to antimicrobial
drugs particularly antibiotics.
Genetic Map of the resistance plasmid R100
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Plasmids must replicate or they will be lost by dilution as their host cell grows and
divides.
Plasmid replication. There is substantial variation in plasmid replication strategies.
Theta-replication
Bidirectional
Unidirectional
One or multiple origins
Rolling circle replication
One strand is replicated starting from a nick in the DNA that provides the 3’-OH.
The double-stranded origin (dso) forms a cruciform structure that binds the plasmidencoded Rep protein. Rep nicks the origin and binds covalently to the 5’ phosphate of one
strand by via a tyrosine residue. Continuous DNA replication begins at the arrow head and
displaces a single parental strand of the plasmid with Rep still bound. Another nick
releases the ssDNA parental strand together with a ds plasmid that consists of 1 parental
and 1 new DNA strain. Replication of the ss begins and the single-stranded origin and
preceeds using an RNA primer and host enzymes.
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Plasmid often carry on them systems that control their copy number
Many different mechanisms are used.
ColE1 plasmid use an antisense RNA to
control copy number. ColE1 replication
starts with transcription across the origin
of replication. While the transcript is still
bound to the DNA template it is cut by
RNAase H to yield the primer for DNA
replication.
Transcription across the origin in the
opposite direction produces an antisense
RNA that binds the pre-primer transcript
upstream from the cut site and prevents
cleavage by RNAaseH to yield the primer
for DNA synthesis. The level of the
antisense RNA (RNA 1) must reach
sufficient concentration to block
replication which requires 20-50 copies of
the plasmid per cell.
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The transfer of DNA between bacterial cells plays and important role in evolution and
is also critical to genetic engineering of bacteria for biotechnology applications.
CONJUGATION. Direct cell-to-cell transfer of DNA (usually conjugative plasmids).
Conjugative plasmids are capable of mediating their own transfer by conjugation. The Fplasmid is the best known conjugative plasmid. The F-plasmid encodes at F-pilus used for
cell-cell attachment and transfer functions (tra) to transfer DNA from cell to cell.
Conjugation (mating) occurs between a cell harboring the F-plasmid (F+) and a cell without
the F-plasmid (F-). Mating between two F+ cells in inhibited by immunity factors.
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An F+ and an F- cell are attached by the F-pilus. The TraN and TraG proteins at the tip of
the pilus bind to the OmpA outer membrane protein of the F- cell. The F-pilus
depolymerizes drawing the cells together. The cell envelopes fuse and a pore is formed by
TraD. A signal is sent through a series of Tra proteins that leads to the binding of TraI
endonuclease/helicase to OriT. TraI nicks the origin and becomes attached to the DNA
after which it acts as a pilot protein guiding the ssDNA through the pore and then binding
to the membrane of the recipient cell. ssDNA continues to move through the pore and the
ssDNA remaining behind in the host is replicated. The ssDNA transferred to the host is
used as a template to make dsDNA. When synthesis completes TraI ligates the double
stranded ends to finish plasmid assembly. The
recipient is now F+.
Hfr (high frequency of recombination): F-factors
can integrate into the chromosome, usually by
homologous recombination between IS elements, to
form and Hfr. Hfrs can transfer DNA starting from
oriT and extending into the chromosome near the
integration site. In some cases nearly the entire
chromosome can be transferred.
F’ (F-prime). F plasmids that have integrated into the chromosome can precisely excise by
homologous recombination to reform the F-plasmid, but that can also excise by illegitimate
recombination between the F and the chromosome of just the chromosome. This latter
case will from an F-plasmid that carries a piece of the chromosome which is known as an F’.
The F’ plasmids are useful in complementation studies.
Complementation
Pheromones
Conjugation by some gram-positive streptococci is mediated by small- diffusible
substances that enhance conjugation- these compounds are called pheromones.
Recipient cells excrete small soluble peptides that induce donor cells to become adherent.
1.
2.
Recipient cell excrete cAD1 a small diffusible oligopeptide “pheromone”: The
recipient takes up cAD1 via an oligopeptide permease.
Once inside the donor cell cAD1 binds to the TraA repressor releasing it from
DNA and derepressing a number of tra genes.
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3.
4.
5.
75
One of the induced genes is the TraE positive regulator which induces asa-1, the
gene encoding an aggregation substance (AS) factor, which is localized on the
donor cell surface
AS factor is a microfibrillar substance that recognizes a binding substance (BS)
present on potential recipients- this binding stimulates conjugal transfer.
Once the plasmid is received the new cell keeps from stimulating reception of
other plasmids by producing an inhibitor of the AD1 oligopeptide pheromone.
TRANSFORMATION
In bacteria transformation is the uptake of DNA from the environment with incorporation
into the host genome. If the molecule is a plasmid, it will be maintained as a plasmid. This
can only occur in a compatible host. If the molecule is a linear fragment of DNA, it must
be incorporated into the recipient genome in order to be stably maintained.
Competence: Only competent bacteria can be transformed. Some bacteria are always
competent, others only become competent in late stationary phase and still other become
competent only after chemical treatments carried out in the lab.
Transformation in Streptococcus- The competent state for transformation is transient
and short.
1. Competence is induced by a competence-stimulating factor (CSF, a 17 amino acid
peptide).
2. Binding of CSF to the membrane receptor stimulates the expression of at least 12
genes.
3. This is a form of cell-density dependent gene regulation (quorum-sensing) where
CSF is active only when it accumulates to a sufficiently high concentration.
4. CSF initiates a signal transduction phosphorelay (com-P) that stimulates expression
of a variety of genes, including CSF synthesis, transport, receptors and an
alternative sigma factor (SigH) that in turns stimulates a variety of operons
involved in the generation of a Translocasome.
5. Translocasome Activity
a. ds DNA binds to the cell surface.
b. ds DNA is nicked by a nuclease and then is degraded into one strand DNA
c. the remaining strand passes through the cell wall and SSB’s DNA binding
proteins attach to the DNA as it enters the cell.
d. the ssDNA is integrated by the process of homologous recombination.
e. The Hex mismatch repair system (related to E. coli methyl-directed mismatch
repair) sometimes removes the newly incorporated ssDNA. If repair occurs
tranformation is not observed, but the repair systems are not perfect.
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Biological role of Transformation
1. A means to acquire DNA as a food source. This makes sense for streptococcus
which can take-up any type of DNA but some species exhibit specificity in DNA
uptake- only DNA from the same species.
2. A role in DNA repair- lysed cells provide a source of DNA that can be used to
repair lethal mutations
3. For exploring the fitness landscape- horizontally acquired genes may increase
fitness (survival) then these genes will be retained and the more fit strain will
ultimately dominate the population
Transformation in Streptococcus
Competence in gram negative bacteria
Neisseria: Neisseria is naturally competent during stationary phase and a CSF is
not needed to induce competence. In G- bacteria, DNA from the environment must also
pass the outer membrane (not present in G+ bacteria) as well as the peptidoglycan layer
and the inner membrane. Neisseria uses a Type IV pilus to take up DNA. Type IV pili
polymerase and deploymerise to move like a piston that is though to drag membrane-bound
DNA into the cell. Before the pilus goes into action the DNA must bind the outer
membrane and interact with an unknown protein the recognizes GCCGTCTGAA. This
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indicates the DNA is from Neisseria which randomly contains this sequence dispersed
throughout its genome.
Heamophilus becomes competent when shifted to starvation conditions. In
Heamophilus specialized vesicles bud from the surface and recognize the sequence
AAGTGCGGTCA within environmental DNA and mediate its uptake. The AAGTGCGGTCA
recognition sequence is present a 4 kb intervals in the Heamophilus genome.
The fact that Neisseria and Heamopilus recognize specific DNA sequence suggest DNA
uptake is not for food.
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BACTERIOPHAGE T4, LAMBDA
There are far more bacterial viruses on Earth than any other
biological organisms. They affect the ecology of all habitats
and the evolution of all organisms.
Viruses are also important in genetic engineering and have
potential application to microbial control.
T4 is a bacteriophage (bacterial virus) that infects E. coli.
Viruses often infect a cell and lyse releasing more viruses.
Infection cycle (phage T4)
Bacterial viruses inject their DNA into the host bacterium. They take over the host
biosynthetic machinery to express their own genes. New virus particle generally selfassemble. The host cycle is lysed to release about 100 viruses.
1. Attachment
1. Phages require receptors for attachment- receptor specificity defines hostrange.
2.
Receptors include: flagella, pili, LPS, proteins and teichoic acids. Their absence is
often the basis of host resistance (e.g. a pili-minus mutant)
2. Penetration
1. T4 uses its complex tail structures for penetration. Its tail is composed of a
hollow tube surrounded by a sheath and a baseplate attached to several tail
fibers.
2. Following attachment, autolysin forms a hole in the outer and cytoplasmic
membrane. The tail sheath contracts, the central tube pushes through the hole in
the membrane, and then DNA is injected through the tail tube into the host cell.
3. Expression of phage genes (usually in a temporal order: early, middle, late).
A. Early and middle genes. The host RNA polymerase begins to synthesize phage mRNA
within 2 minutes after infection. This mRNA and others direct the synthesis of early
proteins that:
a. degrade host DNA to stop host gene expression and provide material for phage
DNA synthesis.
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b. modify host RNA polymerases to recognize phage rather than bacterial promoters.
c. synthesize a unique base, hydroxymethylcytosine (HMC), which is contained in the
T4 phage DNA, to protect T4 DNA from attack by the E. coli restriction system.
d. Phage Nucleic Acid Replication. T4 DNA replication requires many proteins as well
as an available pool of the HMC nucleotides. Up to 300 phage genomes may be replicated
per infected host cell.
e. regulatory proteins that induce the late genes
B. Late genes (late mRNA)
After DNA replication. Functions of these late proteins include:
a. Phage structural proteins (e.g. protomers/capsomeres)
b. Proteins that help with phage assembly without becoming part of the
virion structure
c. Proteins involved in cell lysis and phage release
4. Assembly
Nucleic acids (the genome) and protein subunits (and membrane components in
enveloped viruses) are assembled into new virions. Although some phage components
require special enzymes to assist in assembly, most are assembled by self-assembly
mechanisms.
5.
Release of Phage
a. Phage particles are released from the cell by lysing the cell. Lysis occurs with the
aid of several enzymes, including a protein that damages the cytoplasmic
membrane and allows lysozyme to get to the peptidoglycan layer- Lysozyme
degrades peptidoglycan.
b.
Each of the virions can then infect another cell. This is a typical lytic cell cycle.
c.
Some phage are released without lysing their host cell, such as filamentous phage.
First their coat proteins inserted into the cytoplasmic membrane, and then
the coat assembles around the phage DNA as it is secreted through the
cytoplasmic membrane. In this way, new phage is continually released by a
secretory process.
T4-Phage Infection time-line
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Phage Lambda () a temperate phage. Most of the “T” phages (such as T4) of E. coli are
lytic phages. A temperate phage can lyse the host or integrate into the host genome
and replicate along with the host for a time and later initiate the lytic cycle.
Definition of a temperate phage.
Lysogeny
Lysogen
Prophage
Lysogen induction
Lambda phage
Plaques
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Lambda life cycle
Attachment Lambda attaches to the outer membrane protein LamB which is porin that
assists in the uptake of small hydrophilic molecules as well as maltose and maltodextins.
Penetration. The lambda genome (dsDNA) is injected into E. coli. Following injection the
lambda genome circularizes via cos sites.
Expression of viral genes. A very complex system regulates the expression of lambda
genes and determines whether lysis or lysogeny will result.
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DNA replication in lambda.
Many viruses use both theta and rolling circle replication.
The lambda P and O proteins are used for replication together with host replication
enzymes. After injection, lambda circularizes via its cos sites. It then undergoes several
rounds of theta replication (bidirectional) after which it switches to rolling circle
replication. Rolling circle replication produces a long genome concatemer that is packaged
into assembled phage heads. Then a terminase cuts the concatemer to form the cos sites.
The P protine helps with initiation and O recruits the DnaB helicase.
Integration of lambda into the E. coli chromosome.
Lambda integration is a site-specific recombination between the lambda att site (attP) and
the E. coli att site (attB).The attB site is located on the E. coli chromosome between the
gal and bio genes.
E. coli integration host factor (IHF) is also required for integration of lambda. It is
needed to bend the DNA. Recombination occurs within the cores sequence but also
requires 80-150 bp of flanking DNA
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attP core sequence = tCAGCTTt tTtatAc tAAGTTGg
attB core sequence = cCTGCTTt tTtatAc tAACTTGa
Induction of lambda. Induction the process by which a prophage is induced to begin lytic
growth.
Exposure to high levels of UV light induce lambda. Exposure to UV light activates RecA
which induces autoproteolysis in cI. This in turn results in transcription from PR and PL
and lysis.
After excision, the sib region is transcribed as part of the int message. This region codes
for an RNA stem loop that is susceptible to RNAase II cleavage and destabilized this int
message leading to lower levels of the Int protein which favors the excised state.
Evolutionary advantages of lysogeny: Most bacteriophages are temperate.
1. Under conditions of nutrient deprivation, host cells degrade their own mRNA and
protein and become dormant. By entering into lysogeny, a temperate phage can
“ride out the bad times rather than lysing the cell and not having any metabolically
active bacteria to infect”.
2.
Phage conversion. Temperate phages can induce phenotypic changes in the host
cell that are not related to the completion of their life cycle.
a. Infection with the epsilon phage alters the LPS layer of Salmonella; this
eliminates surface phage receptors and prevents infection of the lysogen
by another epsilon phage.
b.
The diptheria toxin thought to be produced by the bacterium
Cornybacterium diptheriae is not really produced by the bacterium but rather by a
prophage in the bacterium.
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CRISPR
Clustered regularly interspaced short palindromic repeats (CRISPR) sequences.
CRISPR sequences are part of a system of acquired immunity that bacteria use to
defend themselves against viruses mainly but also other types of invading DNA. They
are found in >40% of bacterial genomes and >90% of archaeal genomes.
CRISPR loci were originally discovered as a series of short repeated sequences separated
by highly variable host derived sequences as well as various CRISPR-associated genes. The
repeats and spacers vary from 23-47 bp and 21-72 bp, respectively.
Protospacer. The highly variable sequences, derived from invading DNA, that are part of
CRISPR loci. They are cut out of invading DNA where they reside next to PAM sequences.
CRISPR-associated sequences (cas, csn and cse) are used for CRISPR immunity. They
encode proteins used by the CRISPR system.
crRNA. (CRISPR RNA). This RNA derived from captured invading DNA. The crRNA is used
to target invading DNA by complementary base pairing.
tracrRNA. (Transactivating CRISPR RNA) This RNA is needed for maturation of the
crRNA in some CRISPR systems.
Cas9. A nuclease that can cut DNA to make a double-stranded break. Cas9 is only active
after forming a complex with crRNA that will guide it to the invading DNA by
complementary base-pairing..
PAM sequences. (protospace adjacent motif) PAM is a short sequence (such as NGG) the
must be in the target DNA for Cas9 to cut. PAMs are a component of the invading virus or
plasmid, but are not a component of the bacterial genome. This prevents CRISPR from
cutting at the CRISPR locus.
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CRISPRS AND TARGETED GENOME EDITING
Genome editing is critical in research and biotechnology.
CRISPR genome editing is an easy method to make specific mutations in a wide variety of
eukaryotic cell types as well as organisms that have traditionally been challenging to
manipulate genetically. Hence, CRISPRs are a major advance in genetic engineering.
CRISPRs are used to introduce double-stranded breaks at specific sites in a genome. Then,
these breaks can be repaired in a way that leaves and insertion, deletion or a desired base
change behind.
Break repair systems
In eukaryotes, double-stranded breaks are often repaired by non-homologous end-joining
(NHEJ) which leads to insertions or deletions at the break site. This type of mutation will
almost always inactivate a gene.
Alternatively, if a donor molecule is available the double-stranded break will be fixed by
homology-directed repair (occurs in both prokaryotes and eukaryotes).
Hence, if a donor DNA molecule is introduced into a cell that has a double-stranded
break at the desired site a wide variety of desired mutations can be made.
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The donor DNA can be chemically synthesized at low cost and is introduced into the host
cell using standard methods.
The CRISPR system is used to introduce double-stranding breaks at desired sites in a
genome.
The Cas9 nuclease and a guide RNA are introduced into the cell whose genome will be
edited.
The guide RNA (a fusion of crRNA and tracrRNA) is synthesized (using standard methods)
such that the 5' 20 nucleotides are complementary to the desired target site (which will
also have an adjacent PAM sequence). The PAM sequence be present in the target for Cas9
to cleave DNA.
The guide RNA complexes with Cas9 which recognizes the target by complementary basepairing and cleave the target for from a double-stranded break.
The brake can be repaired by a donor DNA (not shown) to produce the desired mutation.
The donor DNA can easily be introduced into the host cell by various methods.
Abbreviations: RNA (CRISPR RNA) and tracrRNA (transactivating CRISPR RNA)
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CLASS 12 AND 13 REGULATION OF GENE EXPRESSION
As a rule, bacteria express genes only when they are needed. This saves the high
energy cost of protein synthesis. For example, E. coli only expresses the genes needed for
degrading lactose as a carbon source when lactose is present in its environment. In this
case, E. coli makes energy and biosynthetic building blocks from the lactose. When lactose
is absent the lac genes are not expressed. Keeping these genes off when lactose is absent
saves the ATP that would be needed for their transcription, translation. Under any given
condition E. coli expresses about 2000/~4400 genes in its genome.
In general, gene for a biosynthetic pathway are repressed when the pathway endproduct is available and genes for a catabolic pathway are induced when the substrate
of the pathway is available. For example, when the amino acid tryptophan (W) is present
in the environment the genes for W biosynthesis are repressed (The W needed by the cell
for protein synthesis, etc. is taken up from the environment). When the sugar disaccharide
lactose is present the genes for lactose catabolism are induced otherwise they are off.
Examples of gene induction and repression.
Bacteria often regulate gene expression in response to small molecules such as amino
acids, sugars, enzyme cofactors, and other organic and inorganic compounds needed for
growth.
A small molecule used to regulate gene expression is called an effector molecule, if it
induces gene expression it is called an inducer if it represses gene expression it is called a
corepressor.
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Small molecules regulate transcription by binding to regulatory proteins (aka
transcription factors) and changing their binding affinity for specific DNA sites
usually near the promoter of the genes to be regulated.
Bound transcription factors either induce are repress gene expression by interacting
with RNA polymerase.
Repressor proteins repress transcription when bound to DNA. In general, repressor
proteins bind to an operator (a short DNA sequence) near the promoter (the site where
RNA polymerase binds DNA to start transcription) and block the binding of RNA
polymerase which, of course, prevents transcription. The binding of repressors to DNA is
often controlled by small molecules as described above.
Activator proteins activate transcription when bound to DNA. In general, activator
proteins bind to an activator binding site (a short DNA sequence) upstream of the
promoter (the site where RNA polymerase binds DNA to start transcription) and enhance
the binding of RNA polymerase or help RNA polymerase initiate transcription (form an
open complex). The binding of activators to DNA is often controlled by small molecules as
described above.
Dual function regulatory proteins. Many regulatory proteins can act as either activators
or repressors depending on where they bind DNA.
A given regulatory protein will only regulate specific genes usually those located near its
cognate DNA-binding site.
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Small molecules in conjunction with regulatory proteins can regulate transcription in
four general ways. All the way you can think of based on the way they work.
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Activator proteins may work at a distance
via DNA bending although action at a
distance is less common in bacteria.
Transcriptional control in bacteria is generally more complex than binding of a
transcription factor to a single site.
The arabinose operon (ara).
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GLOBAL REGULATION. P223
Global regulatory systems are used to control of many genes in response to a
stimulus. This is often needed when a major environmental change occurs such as a change
from rich to minimal medium, from aerobic to anaerobic growth, heat shock, acid shock,
starvation, sporulation etc.
Catabolite repression (catabolite control). P 201
In E. coli, catabolite repression is a global regulatory system that allow glucose to be
consumed in preference to many other carbon sources. When glucose is present the
levels of cAMP in the cytoplasm are low. Many genes for carbon source degradation require
high cAMP for expression. cAMP is an effector molecular the binds the catabolite
receptor protein (CRP) also known as CAP. In many cases, the CRP-cAMP complex is needed
along with a carbon-source-specific regulatory protein to activate transcription. For
example, induction of genes for arabinose degradation requires AraC regulatory protein,
cAMP and CRP.
Glucose regulates cAMP levels mainly via the
phosphoenolpyruvate phosphotransferase
(PTS) system for glucose uptake. Enzyme
IIAglu is a component of the PTS system that
transfers a phosphate group to glucose as it
is transported inside the cell. When enzyme
IIAglu is phosphorylated it activates
adenylate cylase to convert ATP to cAMP
and cAMP levels rise. When enzyme IIAglu
dephosphorylated it binds to and inactivates
adenylate cyclase keeping cAMP levels low.
The presence of glucose keeps enzyme IIAglu
dephosporylated because it is constantly
transferring phosphate to glucose.
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Two-component regulatory systems.
Two component systems are a widely used family of regulatory proteins used to
control gene expression.
Two-component regulatory systems usually consist of a sensor kinase and a response
regulator hence their name. In addition, many also have a phosphatase that
dephosporylates the response regulator.
The sensor kinase will autophosphylate at a histidines residue in response to an effector.
The phosphate is transferred from the sensor kinase to an aspartate residue on the
response regulator. Down regulation usually occurs via a phosphatase, which may be part of
the sensor kinase or a separate protein, that removes the phosphate from the response
regulator. As you might guess variations apply: phosphorylation can increase or decrease
DNA binding affinity and DNA binding can induce or repress gene expression. Although
most sensor kinases are transmembrane some are cytoplasmic.
Most sensor kinases have an input domain and a
transmitter domain containing the conserved
histidines phosphorylation site. Most response
regulators have a receiver domain that contains
the aspartate phosphorylation site and a DNAbinding domain. Some sensor kinases have
multiple domains.
Two component systems are numerous and widespread in bacteria. They can be
relatively easily identified based on amino acid sequence similarity shared by the sensor
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kinase input domain and the response regulator receiver domain. E. coli is estimated to
have 32.
Some two-component systems found in E. coli
Cross-talk among two-component regulatory systems.
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Transport systems may also contribute to gene regulation.
The Pho Regulon. P. 232-233
The pho regulon is composed of genes that are regulated in response to phosphate
starvation. A number of genes for phosphate acquisition are induced including a highaffinity phosphate transport system, PST, that also participates in phosphate regulation.
The main players in the system are the following:
PhoR, PhoB, phoU, PstB, the pho box (CTTTTCATAAAACTGTCA)
Fast phosphate uptake stimulates PstB to interact with PhoR and inhibit phosphorylation.
In addition PhoR will then interact with PhoU to dephosporylate PhoB
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Quorum-sensing. P. 234
Many bacteria us quorum sensing to regulate gene expression in response to celldensity.
In quorum sensing, gene regulation occurs when a freely diffusible molecule (freely
diffusible across cell membranes) accumulates to a particular threshold level.
Acyl homoserine lactone (AHL) is a common quorum sensing molecule in many bacteria.
expression
Different organisms produce
different acyl homoserine lactones
(AHL’s), the difference is in the
acyl chain, as well as other types of
molecules.
Quorum sensing can activate
transcription of single operons or
genes, or multiple operons.
It is estimated that expression of
1-4% to 10% of P. aeruginosa genes
are quorum sensing controlled, some
of the genes are in operons organized over large patches of the genome.
Functions that are regulated by quorum sensing:
Conjugal transfer of plasmids A. tumefaciens - induces Ti (tumor inducing) plasmid
conjugal transfer
Extracellular polysaccharide production; Capsular polysaccharide production
Biofilm initiation
Nodule number
Virulence factors (proteases, extracellular enzymes (chitinases, pectinases)
Antibiotic production (for biological control and virulence of a pathogen)
Stationary phase growth and survival
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The lux quorum-sensing system is used to control luminescence in Vibrio
Cross-species quorum signaling can occur.
These signals may function not only within a population, but also between populations, since
the signal produced by many organisms are recognized by others, albeit they may not be
quite as efficient at inducing genes. For example, a P. aureofaciens mutant deficient in
signal production was constructed; it was found that phenazine production could be
restored by growth in the presence of about 8% of 700 rhizosphere isolates tested.
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Nearly all bacteria use alternative sigma factors to regulate gene expression. p. 220
RNA polymerase consists of 5 subunits, α2ββ’σ. The σ (sigma) subunit dissociates from the
core polymerase shortly after transcription begins, then reassociates with the core
enzyme after it is released from the template. The sigma factor is required for promoter
recognition and alternative sigma factors can be used to recognize distinct sets of genes.
These sets can be considered regulons: a set of operons that are controlled by the same
regulatory protein(s). For example, in response to an increase in temperature, the
concentration of a particular sigma factor increases and directs the RNA polymerase to
bind to the promoter of particular genes and operons, resulting in the synthesis of heatshock proteins.
Sigma70 is absolutely essential for growth (genes of the protein synthesizing system –
substrate uptake, DNA replication, ribosome synthesis) whereas the other sigma factors
are not essential
Sigma38 or S – RpoS. not essential and regulates genes involved in stress management and
maintenance
Sigma32 or H, RpoH, heat-shock protein genes, deal with the presence of unfolded,
misfolded, damaged or aggregated cytosolic proteins
Sigma54 (rpoN)- deal with nutrient limitations such as nitrogen assimilation and/or fixation,
utilization of alternative carbon sources- does not change in abundance during entry into
the stationary phase
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Control of Alternative Sigma factor Availability
Gene Induction- de novo synthesis (new synthesis) sigma factor expressed in response to
a signal
mRNA half-life/stability- heat shock- rpoH mRNA forms secondary structure that melts
under high Temp
Proteolysis- targeted degradation
There is little transcriptional regulation of the rpoS gene, and the rpoS transcript is
always present in the cell- even during exponential growth. What occurs during the onset
of stationary phase is an increase in translation of the rpoS message and decreased
proteolysis of the sigmaS protein.
SigmaS-is degraded by the ClpPX protease complex- under rapidly growing conditions ClpXP
rapidly degrades the SigmaS- protein but as growth slows ClpXP activity diminishes.
Competition for RNApol- SigmaS has a much greater affinity for RNApol and therefore
displaces Sigma70
Anti-sigma factorsProteins that inhibit sigma-factor activity are called anti-sigma factors. These negative
regulatory proteins interact directly with their cognate sigma-factor, preventing them
from associating with core RNApol.
Pseudomonads can have as many as 18 or more extracytoplasmic function (ECF) sigma
factors that respond to external signals by means of a membrane-localized anti-sigma
factor.
ECF sigma factors- many have membrane bound anti-sigma factors that bind the
sigma factor and disruption of membrane integrity results in release of the sigma
factor and altered patterns of gene expression, presumably targeted to the stress
that altered/disturbed membrane integrity.
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Bacteria also use regulatory RNA molecules to control gene expression.
The CsrAB system uses a regulatory RNA that titrates the regulator. P 222
One of the main function of the CsrAB system is
to regulate carbon storage genes, glycogen
biosynthesis. CsrA is a small 61 amino acid
protein and CsrB is a RNA molecule that contains
18 repeats of the sequence CAGGA(UCA)G that
bind CsrA.
CsrA can bind to target mRNA blocking the RBS
site and destabilizing the message resulting in
reduced gene expression. CsrB sequesters CsrA
by binding it.
Many of the details of how this system works
are still uncertain, but under high carbon csrB is
expressed and glycogen biosynthesis goes up.
Riboswitches.
Cis-acting RNA regulatory elements that block translation/transcription.
Riboswitches are usually 5”-UTRs that bind a metabolite and repress
transcription/translation.
When translation is blocked transcription is blocked by polarity.
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Antisense RNA.
A small regulatory RNA, called antisense RNA, regulates gene activity by binding to a
complementary DNA sequence and preventing translation.
dsRNA is targeted for degradation by RNases
Example: Staphylococcus aureus produces a sRNA called RNAIII (514 nt) that controls a
large number of virulence genes. The 3” end forms a hairpin loops that interacts with the
hairpin loops at the 5’ end of target mRNAs, such as sa1000 (fibrinogen binding
protein)/spa Staphylococcus protein A), frequently at the RBS (SD)- prevent ribosome
binding
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Translational repression-growth rate regulation of
ribosome synthesis. Ribosomal proteins are synthesized from
about 16 transciptional units. In most cases, one or more
protein from each unit represses translation of its own
message.
Many ribosomal protein bind to rRNA, or regulation
translation by binding to similar RNA structure in their own
messages.
Predicted secondary structure of S4 binding sites on rRNA
and S4 mRNA
-----------------------------------------------------------------------------------Definitions and terms for gene regulation
Gene - A sequence of DNA coding for a polypeptide (or rRNA or tRNA).
Operon - A sequence of DNA containing one or more structural genes and one promoter.
Operator - a region of the operon involved in regulating expression of the operon
Regulon - a set of operons that are controlled by the same regulatory protein(s)
Stimulon – all the genes, operons, regulons, modulons that respond to a common
environmental stress
Constitutive – genes that are always expressed.
Inducible - genes whose expression can be induced (increased) in response to a signal.
Repressible - genes whose expression can be repressed (reduced) in response to a signal.
Derepression – induction of gene expression by relief of repression.
Effector molecules: small molecules (inducers and corepressors) that affect gene
expression
Inducer – a small molecule that induces gene expression
Corepressors – a small molecule that represses gene expression.
Repressor protein – a transcription factor (protein) that represses transcription when
bound to DNA.
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Activator protein – a transcription factor (protein) that activates transcription when
bound to DNA.
Negative control – when a repressor protein prevents transcription
Positive control – when an activator protein promotes transcription
Quorum sensing – A regulatory system that requires a certain density of cells of the same
or similar species be present before the regulatory events occur
Two-component regulatory system – a system containing two proteins: a sensor kinase and
a response regulator. The sensor kinase is phosphorylated in response to an external
signal and transfers the phosphoryl group to the response regulator, which controls
transcription of a gene or genes
Stringent response – a global regulatory control activated by amino acid starvation
Alarmone- intracellular signal molecules that accumulate during stress (eg., cAMP or
ppGpp)
Attenuation - regulation based on controlling the continuation of transcription
-----------------------------------------------------------------------------------------