• Define the terms genome, genotype, phenotype, chromosome and nucleoid in the context of
the cell structure and characteristics of prokaryotes.
Phenotype – physical features and functional traits of an organism, a reflection of the proteins
possessed at any point in time
Gene – unit of genetic information, segment of DNA which possesses info specifying the sequence of AA
in a protein or the sequence of nt in a stable RNA molecule
Genotype – particular set of genes in the organism’s genome
Genome – total physical DNA that specifies an organism’s characteristics
• Explain the importance of proteins and diversity of protein function to life.
Structure, catalysis, regulation, transport, sensing/signalling, force-generation
• Describe how genes and proteins are named.
Gene: rpsL or rpsL
Protein: RpsL
• Explain why and how chromosomal DNA is folded and compacted in Bacteria and Archaea.
Different packing in Bac and Archaea. In Bacteria, small positively charged polyamines used to
neutralize negatively charged phosphates of DNA reducing repulsive forces and helping to compact the
DNA.
Also, supercoiling (like a telephone cord twisting). Bacteria produce enzymes that can twice DNA into
supercoils.
The boundaries of each loop of a folded chromosome has anchoring proteins called histone-like
proteins. Histones are also positively charged.
In Archaea, DNA is also supercoiled and some Archaea use histone proteins (similar to Euk) to compact
their DNA. Nucleosome.
• Describe the nature of the prokaryotic nucleoid highlighting the differences between Bacteria and
Archaea as well as the unique features of the nucleoid in some Planctomyces
In Bacteria and Archaea, chromosome refers to the DNA component of the nucleoid. Most prok possess
singular circular chromosome, some possess single linear chromosome, some possess segmented
genome. Difference not understood why. Chromosome refers to chromosome and plasmids and
prophage.
• Explain the structural and functional relationships between the linear structure of a prokaryotic
protein encoding gene, a mRNA and a protein.
• Explain what is meant by gene expression and why genes need to be expressed to have an effect on an
organism’s characteristics
Genes need to be expressed  mRNA  protein (or tRNA or rRNA).
Genotype determines phenotype but to affect the phenotype, the proteins specified by the genes need
to be synthesized (from the genes). When the protein is made, the gene is said to be expressed. Gene
expression is the process of transcription (and then translation if it was mRNA). Central dogma. Use
RNAP and ribosomes.
Gene expression depends on many proteins which have the ability to bind to DNA or RNA. Nucleic acid
binding proteins tend to be specific. Some only bind to ssDNA, or dsDNA or RNA, etc.
• Distinguish between transcription and translation.
• Explain the process of bacterial gene expression from the standpoints of initiation, elongation and
termination during the processes of transcription and translation.
Prokaryotic mRNA is made out of a 5’ UTR containing the ribosome binding site (5 nt), 5 – 9 spacer nt,
the initiation/start codon AUG (5’ to 3’), the coding region, the translation termination or stop codon
(UGA, UAA or UAG) and 3’ UTR containing the transcription terminator, a region of dsDNA. Region b/t 5’
and 3’ UTR called coding region b/c consists of codons.
Nt sequence of mRNA is used by ribosomes as guide to link AA together in a particular sequence
resulting in a unique protein. Ribosomes specifically bind to the mRNA at the ribosome binding site to
begin translation. Rbs are about 5 bases in size. There are different rbs.
After binding at the rbs, with tRNA, ribosomes move along mRNA from 5’ to 3’ joining AA via peptide
linkages. Translation can occur at the same time as transcription. Nucleotides are read by the
ribosomes and tRNA.
The vast majority of prokaryotic proteins begin with AA methionine specified by AUG. Proteins are
synthesized from N terminus to C terminus. That means the 5’ end of the mRNA is for the N terminus.
Proteins can synthesize methionine at other positions, but translation only starts at the AUG suitably
spaced (5-9 nt) from the rbs.
Transcription promoter is the dsDNA region where RNAP initially binds to start transcription. It
therefore defines the beginning of a gene and is about 50 bp. Bacteria have a single RNAP with 4 core
subunits – 2 alpha and 1 beta and 1 beta prime. RNAP exists as a holoenzyme or core enzyme
(different). The core enzyme must associate with a 5th protein called the sigma subunit to form the
holoenzyme. Sigma binds to the promoter first, then RNAP binds and slides along to find the promoter
sequence. When detected, it stops and transcription is initiated as the sigma falls off. There are many
sigma subunits. Control transcription of entire categories of genes by controlling which sigmas are
made.
In Archaea and Euk, RNAP have multiple subunits and none are capable of recognizing promoters.
Instead, transcription factors recognize and bind to sites adjacent to promoters. When the RNAP slides
along the DNA, it stops and initiates transcription when it encounters a bound transcription factor.
Following promoter is the coding region, largest part of gene that specifies order of AA. 1 kb gene has
an average coding region of 900 bp  299 AA. It takes 3 nt pairs (6 nucleotides) in DNA to specify 1
amino acid in a protein.
Following RNAP binding to the promoter, RNAP separates the DNA strands to gain access to the
template strand and moves 3’ to 5’ synthesizing mRNA 5’ to 3’. Enzyme uses nt sequence of template as
a guide for choosing the complementary nt to link together.
Termination.
Transcription termination is poorly understood in Euk and Arc. Only look at Bac now. Two mechanisms
– Rho independent termination and Rho dependent termination.
RNA binding protein called Rho which binds to C-rich sequences in the RNA that is not being translated,
then slides along RNA in 5’ to 3’ direction, towards the RNAP. When it catches up to the RNAP, it pulls
the transcript free of the RNAP and template, releasing it. This happens at sequences that cause RNAP
to pause briefly, thus allowing Rho to catch up. Rho-dependent termination can stop transcription when
the RNAP has moved beyond the end of the last gene in an operon.
Rho-independent termination. RNAP falls off teh template when it is paused over a series of A-U base
pairs (which are relatively weak). Pausing is caused by a stem-loop structure in the RNA. The DNA
sequences that encode rho-independent transcription terminators can be recognized as palindromes.
When the terminator sequence is transcribed into RNA, forms loop – double stranded hairpin like
structure called the transcription terminator. Should be after the last codon.
• State the general differences between the processes of bacterial and archaeal gene expression.
The differences mainly lie in the process of transcription initiation. For bacteria, there is a sigma and the
RNAP core enzyme is made out of 2 alpha, 1 beta and 1 beta prime subunit which all join to form one
enzyme. It joins with the sigma to form the holoenzyme. The sigma is the one responsible for
identifying the promoter region on the dsDNA. The sigma subunit falls off once transcription has
started.
In the Archaea, the RNAP is different. Archaea and Euk have similar transcription initiation methods
(compared to Arc and Bac). In Archaea, the RNAP is made out of several protein subunits and none of
them are similar to sigma. There are transcription factors that bind to the sites adjacent to the
promoters. When RNAP slides along the DNA, it stops and initiates transcription when it encounters the
bound transcription factor.
• Explain why transcription and translation of a gene can occur at the same time in prokaryotes but not
in
eukaryotes and why many copies of the same protein can be simultaneously translated from a single
prokaryotic mRNA.
Nucleus.
• Describe the similarities and differences between prokaryotic protein-encoding genes and those genes
that encode stable rRNA molecules.
Prokaryotes have a substantial number of genes that encode stable RNA. In Bacteria and Archaea, these
genes typically occur in operons. This makes sense because both rRNA and tRNA needed to do protein
synthesis, makes sense to have genes for them in the same operon. Initially RNA products are made
together as one long precursor RNA. Nuclease enzymes cleave the precursor molecule yielding
individual RNA products.
• Explain the structure of operons and their significance.
In Bacteria and Archaea, common to find genes clustered together in the genome if they specify
proteins involved in the same cellular process. These gene clusters are called operons. Genes of
operons are transcribed together as a single mRNA initiating at one promoter, but two or more different
proteins are translated from the single mRNA. Thus on the single mRNA there would be two rbs, two
AUG and two stop codons. But only one transcription terminator after the 2nd stop codon.
• Compare and contrast the expression of genes in an operon composed of protein-encoding genes and
and one composed of stable RNA genes.
• Distinguish between the number of unique protein molecules a prokaryote can synthesize and the
total number of protein molecules that may be present in a prokaryotic cell at any point in time.
Not all proteins are produced due to gene expression – depends on environment and need.
• Using examples explain why different proteins are normally present in a prokaryotic cell in
in a different number of copies.
Proteins needed in difference amounts. RNAP always needed but DNAP not.
• Explain why prokaryotes regulate gene expression.
Different proteins needed in different amounts. Don’t want to waste resources. Don’t want to produce
something that isn’t required. Waste of energy. If waste energy, growth rate will be slowed.
• Distinguish between constitutive and environmentally-regulated genes and gene expression.
• Explain two ways the expression of constitutively-expressed genes is regulated in prokaryotes.
One way of controlling this is through promoter strength. A strong promoter has a nucleotide sequence
that RNAP binds to more strongly than a weak promoter. If it binds more strongly, it is more likely
initiate transcription. A weak promoter tends not to be bound as much, that is – most of the time, RNAP
will not be found bound there so it won’t be transcribing as much. Isn’t all or nothing event – dynamic
equilibrium.
Another way is mRNA stability. If mRNA is less stable, it will tend to be degraded at a higher rate. If it’s
not around for that long after transcription, the protein may not be able to be made as much. As soon
as they are made, mRNAs subject to degradation by ribonucleases (RNases). If more specific protein
needed, more of the mRNA must be transcribed. RNase E is important in the first step of mRNA
degradation. Different mRNAs have different half lives. Some have only a minute some have an hour.
Longer it lasts, more protein can be made. One factor that affects mRNA half life is the nucleotide
sequence of the 5’ UTR. This affects RNase E binding presumably, which initiates the process of
degradation. rRNA is protected by proteins making up the ribosome. Double stranded RNA is usually
not subject to degradation as much. RNase binds to the 5’ UTR and moves 5’ to 3’ making nicks and
therefore equally sized mRNA segments. These segments are further processes by other RNase
enzymes.
• Describe the protein components of the iron acquisition system in E. coli and their function.
Iron is important for nearly all life. Usually insoluble as Iron oxides or hydroxides which are more
difficult to obtain. Pathogenic bacteria face this problem when growing in the body. For example,
Pseudomonas aeruginosa. E. coli is a gram negative bacteria. There is a protein called FepA in the outer
membrane that serves to transport siderophore-Fe(III) complex. There is a protein called FepB which is
a periplasmic protein that carries siderophore-Fe(III) complex from the outer membrane protein FepA to
FepC in the CM. Actually it’s FepD. FepD is a protein in the CM and underneath it is FepC. The FepC
and D transport the siderophore-Fe(III) complex from the periplasm to the cytoplasm where the Fe(III) is
reduced to Fe(II) and then release d from the siderophore – the siderophore can be recycled. ATP is
required for the CM transporter to do its job.
• Explain how the expression of the genes encoding the iron acquisition system in E. coli is
regulated in response to environmental iron and why the regulatory response makes sense.
The genes encoding proteins for iron acquisition are present in several operons in the E. coli
chromosome. For example, FepC, FepD and FepG are in the same operon – they have the same
promoter, and transcription terminator (but the mRNA has several rbs and start codons and stop
codons). The three gene operon is called fepCDG. They are transcribed together as one mRNA from
which the 3 proteins are then translated. Expression of the fepBDG genes is regulated such that the
fepCDG operon is transcribed at high levels only when Fe is limiting in the environment. When sufficient
iron is present in the environment, the fepCDG operon is transcribed at very low levels.
Overlapping the promoter sequence is an operator which is a sequence within the promoter sequence.
The operator is called fur box. fur is a gene. Present in the chromosome of E. coli is regulatory gene fur
which is CONSTITUTIVELY expressed. This is so that the bacterium can ALWAYS be sensing whether
there is iron in the cytoplasm. If there isn’t then it does stuff. fur specifies the protein Fur which is a
regulatory protein that binds to the fur box operator in the promoter region of the fepCDG operon. The
Fur protein therefore recognizes the nt sequence of the fur box operator and can bind there. However,
whether or not it binds actually depends on a signal molecule which is iron which makes sense because
we are talking about acquiring iron and whether we need to make more proteins to get more iron or
not. The iron is the signal molecule that can bind to the specific protein Fur.
When iron binds to the Fur protein, the Fur protein undergoes an comformational change that allows it
to bind strongly to the operator in the promoter region of the fepCDG operon. When it does this, RNAP
cannot bind and transcribe the operon. This makes sense because since there’s iron you don’t need to
make more of the transport proteins to transport more iron in – that would be a waste of resources.
However, when there is no iron present (or little), then Fur protein has a LOWER AFFINITY for the
operator called fur box in the operator of the promoter of fepCDG so RNAP can bind more often (sigma
first) and initiate transcription which makes sense because you would need the proteins to bring in more
iron into the cell transporting the siderophore-Fe(III) complex.
The reason why RNAP cannot bind is because it usually binds to the promoter but the operator overlaps
with the promoter so it’s harder to bind. RNAP actually still transcribes but less when Fur is bound. It
seems like if Fur is not bound to Fe, it cannot bind to the promoter (operator) at all.
A regulon is a set of genes whose expression is regulated in the same way regardless of their location in
the genome. For example, genes for siderophore synthesis and genes for siderophore-Fe transport
constitute a regulon.
• Explain the ways the expression of environmentally-regulated genes is regulated at the level of
transcription initiation in prokaryotes and why a particular regulatory response makes sense.
There are constitutively expressed genes and environmentally expressed genes. Constitutively
expressed genes are always expressed because their protein products are always required. For
example, RNA polymerase. For example the proteins in the ribosome. Transport proteins.
Constitutively expressed genes are still regulated because their degree of expression is fixed at different
levels. This is regulated through mechanisms like promoter strength and mRNA stability. These are
fixed, obviously.
Environmentally regulated genes are expressed at different levels depending on the cell’s environment.
The cell needs to be able to sense the environmental conditions and respond accordingly. They use
numerous mechanisms to sense the environment and translate this information into action. The surface
of the cell has many sensing proteins that monitor osmolarity, pH, etc. They are also sensors for telling
whether other organisms are nearby. Some changes trigger an immediate change in behaviour
WITHOUT the synthesis of any new proteins – chemotaxis for example. Others result in immediate
change in gene expression because new proteins are needed under the new environmental conditions.
This change in gene expression may be sequential – one gene expression changes then another. This is
cell differentiation and it is important that a certain protein is made first before another. In order to
regulate gene expression based on environmental conditions, need to sense environmental conditions
and respond by altering level of gene transcription.
There are four ways that gene expression is controlled at the transcription initiation level when the
gene(s) in question is/are environmentally regulated.
Regulatory proteins can exert positive or negative control. If a regulatory protein exerts positive control,
the gene is said to be positively controlled. This means that when the regulatory protein binds to the
operator for the gene/operon, the gene will be transcribed.
On the other hand, if a regulatory protein that exerts negative control binds to the operator of the
gene/operon, the gene will not be transcribed.
Regulatory proteins that exert positive control are called activator proteins. Ones that exert negative
control are called repressor proteins. The ‘operator’ for activator proteins is actually not part of the
promoter but upstream of it (on the template strand). The operator for repressor proteins is generally
part of the promoter or downstream of the promoter wrt the non-template strand.
Sometimes, operators for the activator proteins are called activator-binding sites. For most operons,
genes specifying regulatory proteins are not part of the operon whose expression they control wtf that’s
obvious. Because if they were they would control themselves. The gene for the regulatory protein is
usually constitutively expressed at low levels.
Whether a regulatory protein binds to the operator (or activator-binding site) depends on another
molecule, an effector molecule aka signal molecule/chemical. In the case of iron acquisition in E.coli, it
was iron. Regulatory proteins are allosteric, meaning they can adopt different shapes and therefore
some shape(s) allow them to bind to the operator or activator-binding site, others don’t. Their shapes
depends on whether they are bound to the effector molecule aka signal molecule or chemical. There
are two different types of effector molecules.
One is an inducer. When the inducer binds to the regulatory protein, then transcription occurs. For an
activator protein, the inducer would cause the protein to bind to the activator-binding site and
transcription would occur at a higher level. For a repressor protein, the inducer would cause it not to
bind to the operator so that transcription can occur.
The other protein is called a corepressor. The corepressor binds to the repressor protein and they bind
to the operator and prevent transcription initiation. For activator proteins, the corepressor is in fact
called an inhibitor because it inhibits transcription by binding to the activator protein and preventing it
from binding to the activator binding site and starting transcription. Usually activator proteins do not
bind strongly to the activator-binding site, or else that would waste lots of resources.
• Using the Aliivibrio fischeri-Eupryma scolopes example, explain how quorum sensing works and its
importance in the life of prokaryotic organisms.
Aliivibrio fischeri is a bacteria that lives in the gut of the squid Eupryma scolopes and they have a
symbiotic relationship. The squid Eupryma scolopes provides the Aliivibrio fischeri with nutrients, a
warm place to live, protection from stuff like protozoa and other things. On the other hand, Aliivibrio
fischeri creates light at night. This makes them look like stars so predators don’t go and eat them, and
they can come out to eat. In the morning the Eupryma scolopes craps so that there’s not too many
bacteria.
Quorum sensing is important because Aliivibrio fischeri will not make light in the absence of other
bacteria. That is, there needs to be a sufficient amount of bacteria first. There’s not much point for only
a single bacterium to make light because that would make very little light. They sense each other by
quorum sensing. It is a type of environmentally regulated gene expression in which gene expression is
regulated by population density. Quorum sensing involves the production of a specific signal molecule
called the autoinducer. The autoinducer can freely diffuse across the cytoplasmic membrane and
outside of the cell and it can enter other cells and induce the expression of certain genes if its
concentration is high enough. If the concentration is high enough then that means that there are many
cells in the area. Each is making the same autoinducer, obviously. Quorum sensing is used to make sure
that there are enough members of the bacteria before it does anything. Otherwise, no effect. Basically
the autoinducer acts as an inducer for an activator protein. So it binds to it, changes its 3D
conformation making it have higher infinity for the activator-binding site on the DNA so that more
mRNA will be transcribed  more proteins produced.
• Using the current information derived from the nucleotide sequencing of prokaryotic genomes
describe
prokaryotic genome diversity in terms of size, gene number, average gene number and gene content
(function, distribution)
About 900 prokaryotic genomes have been sequenced. About 900 are in the progress of being
sequenced. Size is an indicator of the amount of genetic information. Size ranges from 500 to 15,000
kb. Average is 3,700 kb (3,700,000 bases) and median is 3,600 kb. Parasitic genomes are smallest.
Free-living prokaryotic genomes are largest. The average prokaryotic gene is about 1 kb in size. 3’ UTR
and 5’ UTR are about 100 bp so the average coding region is about 900 bp and the average protein
length is 299 AA.
Metabolic and transport genes are typically the most abundant class of genes.
• Using examples explain the correlation between prokaryote genome size and prokaryotic life styles
(eg.
parasitic, free-living etc)
Parasitic genomes like those of mycoplasmas are smallest. Free-living like Sorangium cellulosum are
largest.
• Explain the general principles underlying the use genomic analysis as a way to understand prokaryotes
as well some limitations of this type of analysis.
When we try to identify the function of proteins, we look at the gene that codes for it and compare the
gene sequence with ones we already know of the function that it codes for. Then we say that they are
used for the same thing. This can be inaccurate, and furthermore, not all kinds of proteins are known.
• Solve problems similar to those in the Chapter 3 Questions and Problems handout (VISTA).