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Transcription in Prokaryotes
Transcriptional Control
DNA
RNA
protein
Environmental change
Turn gene(s) on/off
Proteins to deal with new environment
Very important to:1. express genes when needed2. repress genes when not needed3. Conserve energy resources; avoid expressing unnecessary/detrimental genes
Transcriptional Control
DNA
RNA
protein
TranscriptionInitiationElongationTermination
ProcessingCappingSplicingPolyadenylationTurnover
Translation
Protein processing
Many places for control
Prokaryotic Transcription
OperonsGroups of related genes transcribed by the same promoter
Polycistronic RNA
Multiple genes transcribed as ONE TRANSCRIPT
No nucleus, so transcription and translation can occur simultaneously
RNA Structure
Contain ribose instead of deoxyribose
Bases are A,G,C,U, Uracil pairs with adenine Small chemical difference from
DNA, but large structural differences
Single stranded helix Ability to fold into 3D shapes - can
be functional
RNA Structures Vary
RNA more like proteins than DNA:structured domains connected by more flexible domains, leading to different functions
e.g. ribozymes – catalytic RNA
RNA synthesis •
•
RNAP binds, melts DNA
Nucleosides added 5’ 3’
Types of RNA
Messenger RNA (mRNA) – genes that encode proteins
Ribosomal RNA (rRNA) – form the core of ribosomes
Transfer RNA (tRNA) – adaptors that link amino acids to mRNA during translation
Small regulatory RNA – also called non-coding RNA
Transcriptional Control
TranscriptionInitiationElongationTermination
ProcessingCappingSplicingPolyadenylationTurnover
Translation
Protein processing
Control of initiation usually most important.
Initiation
RNA polymerase Transcription factors Promoter DNA
RNAP binding sites Operator – repressor binding Other TF binding sites
Start site of txn is +1
α α β β’σ
Initiation
RNA polymerase 4 core subunits Sigma factor (σ)– determines promoter specificity Core + σ = holoenzyme Binds promoter sequence Catalyzes “open complex” and
transcription of DNA to RNA
RNAP binds specific promoter sequences
Sigma factors recognize consensus-10 and -35 sequences
RNA polymerase promoters
TTGACA TATAAT
Deviation from consensus -10 , -35 sequence leads to weaker gene expression
Bacterial sigma factors
Sigma factors are “transcription factors” Different sigma factors bind RNAP and recognize
specific -10 ,-35 sequences Helps melt DNA to expose transcriptional start
site Most bacteria have major and alternate sigma
factors Promote broad changes in gene expression
E. coli 7 sigma factors B. subtilis 18 sigma factors
Generally, bacteria that live in more varied environments have more sigma factors
Sigma factors
E. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466 T. M. Gruber
Sigma subunit Type of gene controlled # of genes controlled
RpoD Growth/housekeeping ~1000
RpoN N2; stress response ~15
RpoS Stationary phase, virulence ~100
RpoH Heat shock ~40
RpoF Flagella-chemotaxis ~40
RpoE ? ~5
FecI Ferric citrate transport ~5
Extreme heat shock, unfolded proteins
s70
s54
sS
sS
sF
s32
What regulates sigma factors
Number of copies per cell (σ70 more than alternate)
Anti-sigma factors (bind/sequester sigma factors)
Levels of effector molecules Transcription factors
Bacterial RNAP numbers
In log-phase E. coli: ~4000 genes ~2000 core RNA polymerase molecules ~2/3 (1300) are active at a time ~1/3 (650) can bind σ subunits.
Competition of σ for core determines much of a cell’s protein content.
Lac operon control
• Repressor binding prevents RNAP binding promoter
• An activating transcription factor found to be required for full lac operon expression: CAP (or Crp)
lac operon – activator and repressor
CAP = catabolite activator protein
CRP = cAMP receptorprotein
Activating transcription factors
Helix-turn-helix (HTH) bind major groove
of DNA HTH one of
many TF motifs
Crp dimer w/ DNA
Cofactor binding alters conformation
Crp binds cAMP, induces allosteric changes glucose
cAMP
Crp
lac operon
no mRNA
cAMP
Crp
glucose
mRNA
Cooperative binding of Crp and RNAP
Binds more stably than either protein alone
Enhancers• activating regions not necessarily close to RNAP binding site
• NtrC required for RNAP to form open complex
NtrC example:
• NtrC activated by P
• P NtrC binds DNA, forms loop that folds back onto RNAP, initiating transcription• signature of sigma 54
Bacterial promoters
Most bacterial promoters have –35 and –10 elements
Some have UP element Some lack –35 element, but have extended
–10 region
-35 element -10 element (Pribnow box)UP element
pre –10 element
+1
Transcription start
+1
E. coli RNA polymerase composed of 5 subunits:
•Subunits: b, b’, a(2), , and s
•Core enzyme: b, b’, a(2), •Holoenzyme : b, b’, , a(2) and s
•The s subunits give specificity for site of initiation- promoter
Subunit structure of bacterial RNA polymerase
’NTDCTD
NTDCTD
sDNA
Holoenzyme-b’ba2s. Functions in initiation.
Core enzyme-b’ba2. Functions in elongation.
160 kDa
150 kDa
40 kDa
The 3D structure of bacterial RNA polymerase holoenzyme
N-term s1
Inhibition
s2
-10 binding
s3
-10 binding
s4
-35 binding
s factor domains :
s3
The s factors s factors are required for promoter recognition and
transcription initiation in prokaryotes s factors have analogous function as general
transcription factors in eukaryotes A variety of s factors exist in E.coli For expression from most promoters s70 is required For expression from some bacterial promoters one
of other s subunits is needed instead s70 is essential for cell growth in all conditions, while
other sigmas are required for special events, like nitrogen regulation (s54), response to heat shock (s32), sporulation, etc
s
RNA pol
Holoenzyme
Promoter region
Closed complex
Open complex
Promoter escape
Elongation
mRNA s release
-35 -10
The overview of s factor function
The promoter specificity of some s factors in E.coli
s70 TTGACA – 17 bp – TATAATN3-6-A -35 -10 +1
s32 CTTGAAA – 16 bp – CCCCATNTN3-10-T/A -35 -10 +1
s54 GG – N12 – GC/T – 12bp – A -24 -12 +1
The UP element
UP element is an AT rich motif present in some strong (e.g. rRNA) promoters
UP element interacts directly with C-terminal domain of RNA polymerase asubunits
-35 -10 UP +1
s4 s2-3sRNAPa NTD
a CTD
RNAP
Constitutive and inducible promoters
Certain genes are transcribed at all times and circumstances
-Examples – tRNAs, rRNAs, ribosomal proteins, RNA polymerase
-Promoters of those genes are called constitutive Most genes, however, need to be transcribed
only under certain circumstances or periods in cell life cycles
-The promoters of those genes are called inducible and they are subject to up- and down- regulation
Regulation at promoters
Promoters can be regulated by repression and/or activation
Many s70 promoters are controlled both by repression and activation, whereas, for example s54 promoters are controled solely by activation
Cartoon of the transcription cycle
Mechanisms of repression
Repression by steric hindrance Inhibition of transition to open complex Inhibition of promoter clearance Anti-activation Anti-sigma factors
e) Anti-sigma factors An anti-s factor is defined by the ability to prevent
its cognate s factor to compete for core RNA polymerase
Mostly used for s factors, other than s70, for example in life cycle regulation (sporulation, etc)
Some bacteriophages use their own anti-s factors to prevent expression of cellular proteins
-10 -35
RNAP
s
-10 -35
RNAP
santi-s
d) Anti-activation Repressor molecule removes the
activator
weak promoter +1
weak promoter ABS +1
ABS
RNA pol - s
Activator
RNA pol - sActivator
Repressor
Activator binding sequence
Two examples of steric hindrance
Trp repressor Lac repressor
The tryptophan repressor The trp repressor controls the operon for the
synthesis of L-tryptophan in E.coli by a simple negative feedback loop
When enough tryptophane (blue dots) is made, it binds to repressor, which now is able to bind to promoter and block RNA polymerase binding
In the absence of tryptophane the trp repressor (red blob) shows no affinity to promoter (black box) and the RNA polymerase (yellow blob) transcribes the operon
The lac promoter
Lac promoter is widely used in artifical plasmids, designed for protein production
For practical purposes it is easier to use non-hydrolyzable lactose analog – IPTG (isopropyl-b-thiogalactoside) instead of native lactose
A cartoon, ilustrating events upon IPTG binding to lac repressor
As IPTG binds, the DNA binding domains scissor apart
(IPTG)
Mechanisms of activation
a) Regulated recruitment b) Polymerase activation c) Promoter activation
a) Regulated recruitment
Activator “extends” the binding site for RNA polymerase
weak promoter +1 ABS
RNA pol - s
Activator
strong affinity weak affinity
strong or weak affinity
Catabolite Activator Protein: CAP
Activates transcription from more than 150 promoters in E.coli
Upon activation by cAMP (cyclic Adenosine MonoPhosphate), CAP binds to promoter and helps RNAP-s to bind as well
All CAP–dependent promoters have weak –35 sequence, so that RNAP-s is unable to bind the promoter without CAP assistance
Models for Class I and Class II promoter activation
Busby and Ebright, 2000, J. Mol. Biol. 293:199-213
Class I CAP binding sites can be from –62 to –103. CAP interacts with the carboxy terminal domain of the RNAP a-subunit (aCTD)
Class II CAP binding sites usually overlap the –35. CAP interacts with the aCTD, aNTD (N-terminal domain), and the s factor
Model for Class III promoter activation
Activation of Class III promoters requires binding of at least two CAP dimers or at least one CAP dimer and one regulation-specific activator
Interactions can be similar to those of ClassI and/or ClassII promoters, except that each aCTD subunit is making different interactions
AraC – repressor and activator of arabinose
promoter
RNAP-s
RNAP-s
Transcription
+ arabinose ( )
AraC
promoter
DNA binding domain of AraC
RNAP-s54 activation
RNAP-s54 open complex formation requires ATP hydrolysis Activator protein with ATP-ase activity binds to “enhancer”
site about 160 bp upstream from –24 sequence. DNA then gets looped and activator interacts with RNAP-s54 resulting in the open bubble formation upon ATP hydrolysis
s54
s54
ATP ATP+Pi
c) Example of promoter activation: MerR activator family
MerR is an activator that controls genes involved in the response to mercury poisoning
Other MerR family activators (CueR, BmrR, etc) respond to a variety of different toxic compounds such as other heavy metal atoms or drugs
In MerR activated promoters, -10 and –35 regions are separated by 19bp instead of optimal 17bp
DNA-protein interaction assays
Electrophoretic mobility shift assay (EMSA)
DNase I Footprinting
Chromatin immunoprecipitation (ChIP)
EMSA
Radiolabel promoter sequence
Incubate one sample with cell lysates or purified protein and the other without
TF will bind promoter sequence
Run DNA-protein mixture on polyacrylamide gel and visualize w/ audoradiography Free probe
TF-bound probe
EMSA
CovR PcylE
CovR DNA binding proteinBinds to cylE promoter Recognition sequence ‘TATTTTAAT’
DNase I Footprinting
Method to determine where a protein binds a DNA sequence
DNase I footprint
1 -- DNA sequence ladder2 -- DNA sequence ladder3 -- No protein4 -- (+) RNA polymerase5 -- (+) lac repressor
ChIP
Crosslink proteins bound to DNA
Immunoprecipitate lysate for specific transcription factor, RNAP, etc
Analyze DNA bound to protein by PCR
Transcriptional Control
TranscriptionInitiationElongationTermination
ProcessingCappingSplicingPolyadenylationTurnover
Translation
Protein processing
Transcriptional Termination
Bacteria need to end transcription at the end of the gene
2 principle mechanisms of termination in bacteria: Rho-independent (more common) Rho-dependent
Rho-independent termination
• Termination sequence has 2 features:Series of U residuesGC-rich self-complimenting region• GC-rich sequences bind forming stem-loop• Stem-loop causes RNAP to pause• U residues unstable, permit release of RNA chain
Rho-dependent termination Rho is hexameric
protein 70-80 base segment of
RNA wraps around Rho has ATPase activity,
moves along RNA until site of RNAP, unwinds DNA/RNA hybrid
Termination seems to depend on Rho’s ability to “catch up” to RNAP
No obvious sequence similarities, relatively rare
Transcriptional attenuation Attenuator site = DNA sequence where RNAP
chooses between continuing transcription and termination
trp operon 4 RNA regions
for basepairing 2 pairs w/ 1 or 3 3 pairs w/ 2 or 4 Concentration of Trp-tRNATrp determines fate of attenuation At high Trp conc, transcription stops via Rho-independent
Anti-terminationλ phage encode protein that prevents termination
Two Component Systems
Two Component Systems
‘Histidine kinase’ senses environmental changes- autophosphorylates at conserved histidine residue
Response regulator is phosphorylated by activated sensor kinase at conserved aspartate- activates or represses transcription/function
Way for bacteria to sense environmental changes and alter gene expression
Quorum Sensing
Bacteria produce and secrete chemical signal molecules (autoinducers)
Concentration of molecules increases with increasing bacterial density
When critical threshold concentration of molecule is reached, bacteria alter gene expression
Way for communities of bacteria to “talk” to each other
Quorum Sensing in Vibrio fischeri
• at high cell density, V. fischeri express genes for bioluminescence
• LuxI produces autoinducer acyl-homoserine lactone
• AHL diffuses outside of cell
• when AHL reaches critical concentration, it binds LuxR
• activated LuxR bound AHL activates transcription of luminescence genes
Transcription termination
In prokaryotes two types of transcription termination occur – rho indepedent termination and rho dependent termination
In rho independent case, the termination is achieved by a secondary structure of mRNA – RNA stem-loop, followed by an AU rich region
A rho protein is required for rho-dependent termination
Rho independent termination
Attenuation
Regulation of transcription by the behavior of ribosomes
Observed in bacteria, where transcription and translation are tightly coupled
Translation of a mRNA can occur as the mRNA is being synthesized
Attenuation in trp operon
Rho dependent terminationAs polymerase transcribes away from the promoter, rho factor binds to RNA and follows the polymerase
When polymerase reaches some sort of pause site, rho factor catches up with polymerase and unwinds the DNA-RNA hybrid, resulting in release of polymerase
Anti-termination
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