Eukaryotic Expression 1

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Gene Expression
Prokaryotic Gene Transcription
(Cont.)
&
Eukaryotic Transcription
Histones and Chromatin
G & G Pages 336-340, Chapter 29,
9/14/11
Thomas Ryan, Ph.D.
Biochemistry and Molecular Genetics
[email protected]
Prokaryotic Chromosome (E. coli)
• Large circular chromosome 4.6 x 106 bp
• Genome forms a compact
structure called the nucleoid
• DNA organized in 50-100 loops
(domains)
• The ends of loops are
constrained by binding to
protein structure which is in
contact with cell membrane
General Organization of Operons
Operators can be upstream, downstream, or overlapping with
the promoter.
Regulatory proteins that bind to the operator can influence the
access of RNA polymerase to the promoter thereby affecting
the rate of transcription initiation.
Induction and Repression
• Increased synthesis of genes in response to
metabolites is ‘induction’ and the metabolites
are called co-inducers
• Decreased synthesis in response to a metabolite
is termed ‘repression’ and the metabolite a corepressor
• Some substrates induce enzyme synthesis even
though the enzymes can’t metabolize the
substrate - these are ‘gratuitous inducers’ such as IPTG
lac Operon
Both Positive and Negative Regulation
(CRP•cAMP)2
binding
-61.5
RNA Polymerase binding
-35
-10
Repressor binding
+1
mRNA
Multiple control mechanisms are the norm.
lac Operon of E. coli
The lac Operon
Negative regulation
• The structural genes of the lac operon are
controlled by negative regulation
• lacI gene product is the lac repressor
• lacI mutants express the genes needed for
lactose metabolism constitutively
• The lac operator has palindromic DNA
• lac repressor - forms tetramer; DNA binding in
the N-terminal domain; Inducer binding at the
C-terminal domain
lac Operon
lacI gene encodes a repressor
lac Operon
Negative Regulation
•Operon is generally “off”; only fully “on” when lactose is
present and glucose is absent
•When no lactose is present: repressor is bound; inhibiting
transcription elongation through the operon
•When lactose is present: lactose is converted to allolactose,
binds to repressor, causing it to fall off the operator and
allowing transcription of the downstream cistrons to
proceed
Gratuitous Inducers:
Not substrates, but still induce
-Galactoside linkage
CH2OH
HO
O
O
OH
CH2OH
OH
HO
OH
O S
OH
CH2OH
Lactose
C
H
CH3
O OH
OH
CH3
OH
Isopropyl -D-thiogalactoside
(IPTG)
substrate
not a substrate
-galactosidase (LacZ)
CH2OH
O OH
H2O
CH2OH
HO
O
HO
OH
OH
OH
O
OH
OH
O OH
OH
CH2OH
lactose
glucose
CH2OH
-gal -gal
HO
-gal -gal
O OH
OH
OH
Other enzymes in the lac system
lactose permease (LacY)
acetylase (LacA)
galactose
lac Operator Sequence
The operator site is palindromic and lies just downstream
of the transcription initiation site
lac repressor is tetrameric and blocks transcription
elongation by RNAP
Induction of the lac Operon
Binding of -galactosides to the lac repressor induces a cooperative
allosteric change. The inducer:lac repressor complex dissociates from
the operator and transcription of the structural genes occurs.
lac Operon: Catabolite Repression
E. coli can use several sugars as carbon sources but
prefers glucose because requires no energy to take it up
Glucose-sensitive operons: their expression is reduced in
the presence of glucose
Catabolism of glucose inhibits expression of these
operons – called catabolite repression
This mechanism allows for the lac genes to be only
partially induced in the presence of lactose and glucose
Catabolite Activator Protein
Positive Control of the lac Operon
• Some promoters require an accessory protein
to speed transcription
• Catabolite Activator Protein (CAP) is one such
protein
• CAP is a homodimer of 22.5 kD peptides
• N-term binds cAMP; C-term binds DNA
• Binding of CAP-(cAMP)2 to DNA assists
formation of closed promoter complex [also
called CRP (cAMP receptor protein)]
Catabolite Activator Protein
Mechanism of Activation
Glucose starvation activates
adenylyl cyclase increasing
cAMP levels. cAMP acts as a
co-inducer binding to CAP
and activating transcription
by binding to upstream
promoter sequences.
O
Adenine
CH2
O
O
P
O
OH
O
3, 5 cyclic AMP (cAMP)
Catabolite Activator Protein
Upstream promoter
sequence
= cAMP
Binding of CAP to CAP site (e.g. E. coli
lac operon) induces a bend in the DNA
CAP Activation of RNA Polymerase
The trp Operon
Co-repressor Mediated Negative Control
• Encodes a leader sequence and 5 enzymes for tryptophan
biosynthesis
• trp operon is always “on” unless tryptophan levels are
sufficiently high to turn “off”
• Tryptophan is the co-repressor
• Trp repressor binding excludes RNA polymerase from the
promoter
• Trp repressor also regulates trpR and aroH operons and
is itself encoded by the trpR operon. This is autogenous
regulation (autoregulation) - regulation of gene expression
by the product of the gene.
trp Operon
Five structural genes transcribed from the single promoter
repressor
Regulation of the trp Operon by
Repression and Attenuation
Inactive
Active
Attenuation
 The need for tryptophan is tested during translation of the “leader”
sequence (“trpL”) on all mRNA initiated at promoter.
 trp expression is modulated by the tRNA-trp (“charged tRNA”)
available to translate codon UGG. If the ribosomes translate these
codons rapidly, transcription will stop because:
• Different segments in the leader mRNA sequence can pair with
each other to form alternative stem-loop structures.
 If ribosomes “stall” on the trp codons, the RNA structure
formed is not a terminator.
TRANSCRIPTION OF trp OPERON PROCEEDS
 If ribosomes translate the leader and pass the trp codons
quickly, an intrinsic terminator is formed.
TRANSCRIPTION OF trp OPERON TERMINATES
“Pause Hairpin”
“Antiterminator”
“Terminator”
Control of Gene Expression
Gene Expression
Eukaryotic Gene Transcription
Histones and Chromatin
Eukaryotic Vs. Prokaryotic Transcription





Presence of a nucleus means transcription and translation occur in
separate compartments of the cell
Larger genomes (1000X between humans and E. coli)
Chromatin structure in eukaryotes limits accessibility
Three RNA polymerases
Eukaryote pre-mRNAs are subject to extensive post-transcriptional
modification
Genome Sizes of Various Organisms
The human genome contains approximately 1000 times the
amount of DNA compared to E. coli. However, humans have only
20 times as many genes as E. coli. (98.5% of the human genome is
noncoding compare to only 11% of the E. coli genome).
Eukaryotic Chromatin Structure
 The human genome contains 3 x 109 bp per haploid genome.
 If the DNA of all 46 chromosomes from one cell was linked
together, it would measure one meter in length.
 If the DNA in all cells of a single human was linked end to
end, it would stretch to the sun and back.
 How is the DNA packaged into the nucleus such that genes
are still accessible to the transcriptional regulatory proteins
that control their expression?
Characterization of Human Genomic DNA
LINES - Long interspersed nuclear elements (6.1 kb full length)
SINES - Short interspersed nuclear elements (300 bp)
Eukaryotic Chromatin Structure

Each eukaryotic chromosome contains a single
linear supercoiled DNA molecule

Nucleoprotein material of the eukaryotic
chromosome is called chromatin (complex of
DNA, protein and RNA)
Cytoplasm
Nucleus
Nuclear membrane
Nuclear pore
Heterochromatin
Euchromatin
EM of a liver cell: cytoplasm (left) separated from the
nucleus (right) by the nuclear membrane (middle)
Eukaryotic Chromosome Structure
• Euchromatin - comprises most of
the genome, transcriptionally
active, susceptible to DNaseI
digestion
• Heterochromatin - highly
condensed inactive chromatin
located at centromeres and
telomeres (less susceptible to
DNase digestion)
• Centromere - attachment point
for sister chromatids and spindle
fibers
• Telomere - ends of chromosome
Telomeres and Telomerase
Eukaryotic Chromatin Structure




DNA associates with histones proteins to produce
nucleosomes
Nucleosome - the fundamental unit of organization of the
chromatin fiber
Each nucleosome contains a core particle of basic proteins –
histones - which are surrounded by DNA
Electron microscopy shows fiber structure of chromosomes
as “beads on a string”
Histones



Small basic proteins, rich in lysine and arginine
Interact with DNA through electrostatic interactions
Five major types of histones:
• H1, H2A, H2B, H3 and H4
• H2A, H2B, H3 and H4 form a complex of 8
proteins
• DNA is supercoiled around histone octets forming
nucleosomes
• H1 associates with neighboring nucleosomes to
form a more closely packed structure 30nm
solenoid
Histone Structure
(B) Structure of the histone
fold.
(C) Histones 2A and 2B
form a dimer through an
interaction known as the
handshake. Histones H3
and H4 form a dimer
through the same type of
interaction.
Histone Structure
 H2A and H2B
monomers form a
H2A-H2B dimer.
 Similarly, H3 and
H4 monomers
form an H3-H4
dimer.
Histone Structure
• H2A-H2B and H3-H4 dimers bind to form tetramers.
• Two tetramers will come together to form the 8 histone octet
DNA Wraps Around Histone Octomers
 The DNA helix makes 1.65 turns
around the histone octamer.
 146 base pairs of DNA wraps
around each histone octet.
 Histones are among the most
highly conserved eukaryotic
proteins.
For example, the
amino acid sequence of histone
H4 from a pea and a cow differ
at only 2 of the 102 amino acids.
Solenoid Formation: Role of H1 Histone
Formation of the 30nm fiber requires Histone H1 which
is a globular histone that is highly conserved. Histones
are positively charged and this helps to compact the
negatively charged DNA.
Histone Tails
Nucleosomal Core Without DNA
Yellow: H2A
Red: H2B
Blue: H3
Green: H4
 Note ordered structure of
histone amino terminal tails.
These tails can contact neighboring nucleosomes.
 When the tails are acetylated,
interactions between nucleosomes are diminished and
chromatin becomes more
extended or open.
Solenoid
Eukaryotic Chromatin Structure
Electron micrographs of 30nm “Solenoid” structure (top) and
10nm DNA/histone “beads on a string” structure (bottom).
Eukaryotic Chromatin Structure
Looping and Miniband
Eukaryotic Chromatin Structure
•Chromosomes
•attachment of loops of chromatin fibers to
nonhistone protein complexes = scaffold
•Nucleosome fibers condense more into 30
nm chromatin fiber. Predominant form in
interphase nucleus
•Duplex DNA winds around histone octamers
to form nucleosomes = 11 nm histone fiber
•Primary structure of DNA = double helix
= 2nm duplex DNA
Prokaryotes Vs. Eukaryotes
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