Post on 16-Dec-2015
Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination to prevent transcription from proceeding past a terminator to the gene(s) beyond. This is the primary control strategy for bacterial gene expression.
•Molecular Biology Course
Gene expression controls
In eukaryotic cells, processing of the RNA product can also be regulated at the stages of modification, splicing, transport, or stability. In bacteria, an mRNA is in principle available for translation as soon as it is synthesized, and these stages of control are not available.
•Molecular Biology Course
Translation may be regulated, usually at the stages of initiation and termination (like transcription). Regulation of initiation is formally analogous to the regulation of transcription: the circuitry can be drawn in similar terms for regulating initiation of transcription on DNA or initiation of translation on RNA. This regulation will not be detailed in this course.
•Molecular Biology Course
Regulation of Transcription in Prokaryotes
•Molecular Biology Course
Operon directed regulation LAC operon TRP operon
Factor directed regulation
Regulation of Transcription in Prokaryotes
OperonRegulation of Transcription in Prokaryotes
In 1961, Jacob and Monod distinguished between two types of sequences in DNA: sequences that code for trans-acting products; and cis-acting sequences that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA).
Regulation of Transcription in Prokaryotes
A gene is a sequence of DNA that codes for a diffusible product. This product may be protein (as in the case of the majority of genes) or may be RNA (as in the case of bgenes that code for tRNA and rRNA etc.). The crucial feature is that the product diffuses away from its site of synthesis to act elsewhere. Any gene product that is free to diffuse to find its target is described as trans-acting.
The cis-acting sequence applies to any sequence of DNA that is not converted into any other form, but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked.
Operon:Operon: a unit of prokarytoic gene expression which typically includes: 1. Structural genes for enzymes in a specific biosynthetic pathway whose expression is co-ordinately controlled 2. Control elements, such as operator sequence 3. Regulator gene(s) whose products recognize the control elements.
Can be encoded by a gene in another operon
Regulation of Transcription in Prokaryotes
Control element
Structural genes
L1 The Lac OperonL1 The Lac Operon
L2 The Trp OperonL2 The Trp Operon
L3 Transcriptional regulation by alternative σ Factors
L3 Transcriptional regulation by alternative σ Factors
The operon, the lactose operon, the lac repressor, induction, cAMP receptor protein
The trp operon, the trp repressor, the attenuator, leader RNA structure, the leader peptide, attenuation & its importance
Sigma factor, promoter recognition, heat shock, sporulation in B. subtilis, bacteriophage factors
L1L1 The The LacLac OperonOperon L1L1 The The LacLac OperonOperon
1. The operon (done)2. The lactose operon (乳糖操纵
子)3. The lac repressor (乳糖抑制蛋
白)4. Induction (诱导)5. cAMP receptor protein ( CR
P )
Regulation of Transcription in Prokaryotes
The lac operon
Lac repressor
Transcription blocked
Inducer
Activate the Plactranscription CRP + cAMP (gluc
ose repressed)
High level of transcription
(Lactose)
Overview
L1-2L1-2 The Lactose The Lactose OperonOperon L1-2L1-2 The Lactose The Lactose OperonOperon
E. coli can use lactose as a source of carbon. However, the enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source.
L1: The LAC operon
Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements
lacI
Regulatory gene
lacZ lacY lacA DNA
m-RNA
β -GalactosidasePermease
Transacetylase
Protein
Structural GenesCis-acting elements
PlacI Plac Olac
lacY encodes a galactoside permease ( 半乳糖苷渗透酶 )to transport Lactose across the cell wall
lacZ codes for β-galactosidase ( 半乳糖苷酶 ) for lactose hydrolysis
lacA encodes a thiogalactoside transacetylase ( 硫代半乳糖苷转乙酰酶 )for lactose metabolism
L1: The LAC operon
The lacZ, lacY, lacA genes are transcribed from a single (lacZYA) transcription unit under the control of a signal promoter Plac .
LacZYA transcription unit contains an operator site Olac
position between bases -5 and +21 at the 3’-end of Plac
Binds with the lac repressor
L1: The LAC operon
L1-3L1-3 The Lac The Lac repressorrepressor L1-3L1-3 The Lac The Lac repressorrepressor
Regulation of Transcription in Prokaryotes
The repressor is encoded by LacI and active as a tetramer consisting of 4 identical subunits (has a symmetrical structure). It binds to occupies the operator-binding site Olac (28bp, palindromic) and blacks almost all transcription of lacZYA when lack of inducer (such as lactose).
Plac
The repressor and RNA polymerase can bind simultaneously to the lac promoter and operator sites. The lac repressor actually increases the binding of the polymerase to the lac promoter by two orders of magnitude.
Thus, RNA polymerase binds very tightly to Plac but no transcription occur because of the bound repressor
L1: The LAC operon
L1-4L1-4 InductionInduction L1-4L1-4 InductionInduction
When lac repressor binds to the inducer (whose presence is dependent on lactose), it changes conformation and cannot bind to Olac site any more. This allows rapid induction of lacZYA transcription.
L1: The LAC operon
i p o z y a
Very low level of lac mRNA
Absence of lactose
Active
i p o z y a
-Galactosidase
PermeaseTransacetylase
Presence of lactose
Inactive
Lack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA .
Lactose is present, the low basal level of permease allows its uptake, andβ-galactosidase catalyzes the conversion of some lactose to allolactose.
Allolactose acts as an inducer, binding to the lac repressor and inactivate it.
Allolactose causes a change in the conformation of the repressor tetramer , reducing its affinity for the lac operator . The lac operator is removed from the Olac and allows the polymerase to rapidly begin transcription of the lacZYA.
L1: The LAC operon
Lactose (allolactose) is a native inducer to release RNA transcription elongation from Plac .
IPTG, a synthetic inducer, can rapidly simulate transcription of the lac operon structural genes.
IPTG is used to induce the expression of the cloned gene from LacZ promoter in many vectors, such as pUC19.
Ampr
ori
pUC18(3 kb)
MCS (Multiple cloning sites,多科隆位点)
Lac promoter
lacZ’
Gene X
No IPTG, little expression of X geneWith IPTG, efficient expression of X gene.
L1: The LAC operon
L1-5L1-5 cAMP receptor protein cAMP receptor protein (CRP)(CRP)
L1-5L1-5 cAMP receptor protein cAMP receptor protein (CRP)(CRP)
L1: The LAC operon
CRP is a transcriptional activator which is activated by binding to cAMP. However, it is only active when cAMP bound, and cAMP is controlled by glucose. CRP activator mediates the global regulation of gene expression from catabolic operons in response to glucose levels.
L1: The LAC operon
The Plac is a weak promoter, lacking a strong –35 and –10 consensus sequences. High level expression from this promoter requires the activity of the specific activator, CRP.
When glucose is present
The level of cAMP is low in cell, and CRP exists as a dimer which can’t bind to DNA to regulate transcription.
When glucose is absent The level of cAMP increase and CRP bind to cAMP. The CRP-cAMP complex binds to Plac just upstream from the site for RNA polymerase. Induces a 90°bend in DNA which enhances RNA polymerase binding to the promoter and thus the transcription by 50-fold.
CRP-binding site is an inverted repeat.
C A B
Summary
A: RNA polymeraseB: lac repressor C: CRP-cAMP
The CRP (also called CAP) protein can bind at different sites relative to RNA polymerase.
Supp.
L2 The Trp OperonL2 The Trp Operon1. The trp operon2. the trp repressor3. the attenuator4. Leader RNA structure5. The leader peptide6. Attenuation7. Importance of attenuation
Regulation of Transcription in Prokaryotes
L2-1 The Trp OperonL2-1 The Trp Operon
Regulation of Transcription in Prokaryotes
(色氨酸操纵子 ) (色氨酸操纵子 )Bacillus subtilis uses a different regulation m
echanism from what is described here (see the reference of this class).
1. The trp operon encodes five structural genes required for tryptophan synthesis.
2. It encodes a signal transcription ( 7kb, polycistron ) downstream of Otrp.
3. These genes are co-ordinately expressed when tryptophan is in short supply in the cell.
A B C
L2: The trp operon
L2: The trp operon
L2-2 The Trp repressorL2-2 The Trp repressor
Regulation of Transcription in Prokaryotes
(色氨酸阻遏物 ) (色氨酸阻遏物 )
1. Trp repressor is encoded by a separate operon trpR, and specifically interacts with Otrp, a palindrome of 18 bp, and overlaps with the Ptrp sequence between base –21 and +3)
L2: The trp operon
2. The repressor can only bind to the operator Otrp when it is complexed with tryptophan. Therefore, try is a co-repressor and inhibits its own synthesis through end-product inhibition (negative feed-back regulation).
L2: The trp operon3. The repressor reduces transcription ini
tiation by around 70-fold, which is much smaller than the binding of lac repressor.
4. The repressor is a dimer of two subunits which has a structure with a central core and two flexible DNA-reading heads (carboxyl-terminal of each subunit )
L2: The trp operontrpR operon
trp operon
L2-3 The attenuatorL2-3 The attenuator
Regulation of Transcription in Prokaryotes
(衰减子 ) (衰减子 )Repressor does not account for all the reg
ulation: Deletion of a sequence between the opera
tor and trpE gene coding region (attenuator) increase both the basal and the activated (derepressed) levels of transcription.
1. Lies at the end of the transcribed leader sequence that precedes the trpE initiator codon.
2. Is a ρ-independent terminator site (GC-rich palindrome) f0llowed by eight successive U residues.
L2: The trp operon
3. Acts as a highly efficient transcription terminator if the hairpin structure is formed, and only a very short transcipt is synthesized.
L2-4 Leader RNA structure L2-4 Leader RNA structure
Regulation of Transcription in Prokaryotes
(先导 RNA 的结构 ) (先导 RNA 的结构 )
Complementary 3:4 termination of transcription
Complementary 2:3 Elongation of transcription
The leader sequence contains four regions (sequence 1,2,3,4) of complementary sequence that can form different structures
free leader RNA
L2: The trp operon
L2-5 The leader peptide L2-5 The leader peptide
Regulation of Transcription in Prokaryotes
(先导肽 ) (先导肽 )The leader RNA contains an efficient ribosome binding site (RBS) and encodes a 14-amino-acid leader peptide (bases 27-68), Codons 10 and 11 of this peptide encode trp. Thus the availability of trp will affect the translation/ ribosome position, which in turn to regulate transcription termination.
L2-6 Attenuation L2-6 Attenuation
Regulation of Transcription in Prokaryotes
(衰减作用 ) (衰减作用 )Transcription and translation in
bacteria are coupled. Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA, and the attenuation is possible
High trp (attunation)
Lack of trp (proceeding through the whole operon )
Transcription of the trp operon
During transcription of the RNA from trp operon, the RNA Polymerease pauses at the end of sequence 2 (sequences 1 and 2 form a hairpin) until a ribosome began to translate the leader peptide.
L2: The trp operon
High trp
Trp is inserted at the trp codons
Translate to the end of leader message
Ribosome occlude sequence 2
Terminate transcription because 3:4 hairpin formed
L2: The trp operon
Lack of trp
Lack of aminoacyl tRNAphe
Ribosome pause at trp codons , occluding sequence 1
2:3 hairpin (anti-terminator ) forms
Transcription into trpE and beyond
L2: The trp operon
Low Trp
High Trp
L2-7 Importance of attenuation L2-7 Importance of attenuation
Regulation of Transcription in Prokaryotes
(衰减作用的重要性 ) (衰减作用的重要性 )
A typical negative feed-back regulation
Give rise to a 10-fold repression of the trp operon transcription ( 细调) , increasing the regulatory effect up to 700-fold combining the 70-fold repressor effect ( 粗调) .
Faster and more subtle regulation of trp metabolism in bacteria.
Additional: Distinguishing positive and negative control
Additional: Distinguishing positive and negative control
Regulation of Transcription in Prokaryotes
1. Positive and negative control systems are defined by the response of the operon when no regulator protein is present.
2. The characteristics of the two types of control system are mirror images:
• Genes under negative control are expressed unless they are switched off by a repressor protein
• Genes under positive control, expression is possible only when an active regulator protein is present.
To exert a negative control, a trans-acting repressorrepressor either binds to DNA to to prevent RNA polymerase from initiating transcription (inhibits transcription), or binds to mRNA to prevent a ribosome from initiating translation.
In prokaryotes, multiple genes can be controlled coordinately on the transcription level through interaction of repressor with the operator sites. (Lac and trp repressors)
The cis-acting operator/promoter sites are adjacent to the structural genes
Genes are on because RNA polymerase initiates transcription at promoter
Genes are turned off when repressor binds to operator
In positive control, trans-acting activatoactivatorsrs must bind to cis-acting sites in order for RNA polymerase to initiate transcription at the promoter (help transcription), which is opposite to negative control. (CRP activator)
In prokaryotes, multiple genes can be controlled coordinately on the transcription level through interaction of activator with the DNA sites near promoter. (CRP activator)
Gene off by default
Gene turned on by activator
Either positive or negative control could be used to achieve either induction (诱导) or repression (阻遏) by utilizing appropriate interactions between the regulator protein and the small-molecule inducer or corepressor.
Ind
ucti
on
Inducer Inducer
Positive controlNegative control
负控诱导系统 正控诱导系统
Negative controlPositive controlR
ep
ress
ion
Corepressor Corepressor
负控阻遏系统正控阻遏系统
Regulation of Transcription in Prokaryotes
L3 Transcriptional regulation by alternative σ Factors
L3 Transcriptional regulation by alternative σ Factors
1. Sigma factor2. Promoter recognition3. Heat shock 4. Sporulation in B. subtilis5. Bacteriophage factors
L3-1&2: Sigma factor and promoter recognition
L3-1&2: Sigma factor and promoter recognition
Transcriptional regulation by alternative σ Factors
σ factor subunit bound to RNA pol for transcription initiation
Released core enzyme αββ’ω RNA elongation
σ factors is bifunctional protein
Bind to core RNA Pol
Recognize specific promoter sequence
(-35 and –10) in DNA
Transcriptional regulation by alternative σ Factors
factor:
Transcriptional regulation by alternative σ Factors
Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition
E. coli: Heat shock
Sporulation in bacillus subtilis
bacteriophage σfactors
Transcriptional regulation by alternative σ Factors
Different σfactors binding to the same RNA Pol
Confer each of them a new promoter specificity, and allows the diversion of the cell’s basic transcription machinery to the specific transcription of different classes of genes
σ70 factors is the most common σfactor in E. coli under the normal growth condition
Transcriptional regulation by alternative σ Factors
L3-3: Heat shock
L3-3: Heat shock
Transcriptional regulation by alternative σ Factors
The response to heat shock is one example in E. coli where gene expression is altered significantly by the use of different factors.
• Around 17 proteins are specifically expressed in E.coli when the temperature is increased above 37ºC.
• These proteins are expressed through transcription by RNA polymerase using an alternative factor 32 coded by rhoH gene. 32 has its own specific promoter consensus sequences.
Transcriptional regulation by alternative σ Factors
Comparison of the heat-shock 32 and general 70 responsive promoter
Consensus promoter –35 sequence –10 sequence
Standard 70
Heat shock 32
-----------TTGACA----16~18bp---TATAATT-C-C--- CTTGAA--13~15bp--CCCCAT--T
Transcriptional regulation by alternative σ Factors
Heat shock Transiently expression of the 17 heat shock proteins
Increase in temperature is more extremely (50ºC)
Heat shock proteins are the only proteins made in E. coli to maintain its viability
From 37ºC to 42ºC
L3-4: Sporulation in B. subtilis
L3-4: Sporulation in B. subtilis
Transcriptional regulation by alternative σ Factors
• Under non-optimal environmental conditions Bacillus subtilis cells from spores through a basic cell differentiation process involving cell partitioning into mother cell and forespore.
The process of spore formation involves the asymmetrical division of the bacterial cell into two compartments, the forespore, which forms the spore, and the mother cell, which is eventually discarded.
1. Vegetative B. subtilis cell contains a divers
e set of factors 2. Sporulation is regulated by a further set of
factors3. Different factors are specifically acti
ve before cell partition occurs in the forespore and in the mother cell to cross regulate the transcription.
4. Cross-regulation of this compartmentalization permits the forespore and mother cell to tightly co-ordinate the differentiation process.
L3-5: Bacteriophage factors
L3-5: Bacteriophage factors
Transcriptional regulation by alternative σ Factors
• Many bacteriophages synthesize their own factors in order to ‘take over’ the host cell’s transcription machinery by substituting the normal cellular factor and altering the promoter specificity of the RNA polymerase.
1. Many bacteriophages synthesize their own σfactors to endow the host RNA Pol with a different promoter specificity and hence to selectively express their own phage genes .
2. B. subtilis SPO1 phage expresses a cascade of σfactors which allow a defined sequence of expression of different phage genes .
Normal bacterial holonzyme
Express early genes
Encode σfactor for transcription of late
genes
Encode σ28
Express middle genes (gene 34 and 33)