Transcription often is controlled at the stage of initiation. Transcription is not usually...

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.


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.


Regulation of Transcription in Prokaryotes


Operon directed regulation LAC operon TRP operon

Factor directed regulation


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).


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


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


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 ）


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


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).

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


L2-1 The Trp OperonL2-1 The Trp Operon


（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


（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


（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


（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


（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


（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


（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


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




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


factor:


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


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


L3-3: Heat shock

L3-3: Heat shock


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.


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


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


• 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


• 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)

Transcription often is controlled at the stage of initiation. Transcription is not usually...

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