Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences Section L Regulation of Transcription in Prokaryotes L1 The lac operon L2

Section L: Regulation of transcrip. in Prok. Yang Xu, College of Life Sciences

Section L Regulation of Transcription in Prokaryotes

L1 The lac operon L2 The trp operon L3 Transcriptional regulation by alternative factor


L1 The lac operon

• The operon

• The lactose operon

• The lac repressor

• Induction

• cAMP receptor protein

Francois Jacob (44y)

Jacques Monod (55y) (French)

Lac. Operon Theory

Nobelists in Biology in 1965

Concept of mRNA

Francois Jacob

Jacques Monod


The operonDefinition: The operon is a unit of gene expression and regulation

which typically includes:

• Regulator genes: whose products recognize the control-elements, for example a repressor which binds to and regulates an operator sequence.

• Operator sequence: Control elements such as an operator sequence, which is a DNA sequence that regulates transcription of the structural genes.

• Structural genes: The structural genes for encoding proteins.

Structural genesRegulator genes Operator sequence

PlacI lacI Plac Olac lacY lacAlacZ

regulation information


The lactose operonStructural genes: The lactose operon consists of 3 structural genes: lacZ, lacY,

lacA.

Polycistronic mRNA: The three structural genes are encoded in a single transcription unit lacZYA, which has a single promoter Plac, and transcribes a single polycistronic mRNA, but more proteins expressed.


The lac repressorDefinition: The lac I gene encodes the

repressor, which is active as a tetramer of identical subunits. It has a very strong affinity for the lac operator-binding site, Olac, and also has a generally high affinity for DNA.

Structure: The lac operator-binding site consists of 28 bp which is palindromic. This inverted symmetry of the operator matches the inherent symmetry of the lac repressor.

DNA


The lac repressor

Binding: In the absence of lactose, the repressor occupies the operator-binding site. The lac repressor increases the binding of the RNA polymerase to the lac promoter by two order of magnitude. This means that when lac repressor is bound to the Olac, the RNA plo is also likely to be bound to adjacent Plac promoter sequence without move.


PlacI lacI Plac Olac lacY lacAlacZ

Induction process

RNAp

LowLac.

RNApRNApRNApRNApRNAp

mRNA

4. Transcription:

5. Re-Inhibition:

1. No lactose: In the absence of lactose, the lac repressor blocks all but a very low level of transcription of lacZYA.

2. Uptake: When lactose is added to cell, the low level of permease allows its uptake, and galactosidases catalyzes some lactose to allo-lactose.

3. Induction: Allo-lactose acts as an inducer and binds to the lac represser. This causes a change in the conformation of the repressor tetramer, reducing its affinity for lac operator.

RNAp


cAMP receptor protein-IFunction:

• The Plac promoter is not a strong promoter. Plac and related promoters do not have strong -35 sequences and some even have weak -10 consensus sequences.

• For high level transcription, they require the activity of a specific protein called cAMP receptor protein (CRP).

• CRP exists as a dimer which cannot bind to DNA on its own, nor regulate transcription, but which can form CRP-cAMP

CRP-cAMP (cAMP receptor protein) binding site

● CRP-cAMP binds site I

● site I: IR is CRP-cAMP strong binding site （ -70 ~ -50 ）

● site II: is CRP-cAMP weak bingding site （ -50 ~ -40 ）

cooperative effect Promote site II binding

IR

-70

site I

-40

site II

IR

-50

SiteI siteII GC Island -35 -10

● CRP-cAMP + site II promotion RNApol. into -35 sequence

into -10 sequence

starting transcription

RNApol


cAMP receptor protein-II • Normal Condition: The glucose is present in E. coli.

1. Inactivated operon: The E. coli does not require alternative carbon sources such as lactose. Therefore lactose operon is normally inactivated.

2. Inactivated CRP: The glucose can reduce the level of cAMP, therefor the cAMP is not enough for binding CRP.

• Special Condition: When glucose is absent in E. coli culture.1. Activated CRP: The levels of cAMP increased, and CRP

binds to cAMP to form the CRP-cAMP complex which is the activated CRP.

2. Activated operon: The CRP-cAMP complex binds to the Plac just upstream from the site for RNA pol. CRP binding induces a 90º bend in DNA, and to enhance RNA pol binding to the promoter, enhancing transcription by 50-fold.


L2 The trp operon

• The tryptophan operon

• The try repressor

• The attenuator

• Leader RNA structure

• Attenuation

• Importance of attenuation

trp


The tryptophan operon

Structural genes: The trp operon encodes five structural genes whose activity is required for tryptophan synthesis.

Transcript: The operon encodes a single transcription unit which produces a 7 kb transcript which is synthesized downstream from the trp promoter Ptry and trp operator sites Otrp.

Expression: Like many of the operons involved in amino acid biosynthesis, the trp operon has evolved systems for co-ordinated expression of these genes when tryptophan is in short supply in the cell.

Regulation speed: As with the lac operon, the RNA product of this transcription unit is very unstable, enabling bacteria to respond rapidly to changing needs for tryptophan.

trpR P O E D C B A

trpR P O E D C B A

tryptophan

Mechanism

Operator

operon off

operon on

repressor + trp

active repressor

Repressor (inactive )

can not bind

on the O site

(trp absent)


The trp repressorTrp repressor: A gene product of the trpR operon. It

is a dimer of two subunits.

Operator structure: Ptrp is between -21 and +3. The core binding site is a palindrome of 18bp.

Mechanism: The trp repressor can only bind to the operator when it is complexed with tryptophan. The repressor dimer has a structure with a central core and two DNA-reading heads. When tryptophan is bound to the repressor the reading heads are the correct distance apart, and the side chains in the correct conformation, to interact with major grooves of the DNA at Ptrp.

Tryptophan: is the end-product of the enzymes encoded by the trp operon, it acts as a co-repressor and inhibits its own synthesis by end-product inhibition. The repressor reduces transcription initiation by around 70-fold.

trp



The attenuatorBackground: At first, it was thought the repressor was responsible

for all of the transcriptional regulation of the trp operon. And then, it was observed that the deletion of a sequence between the operator and the trpE resulted in an increase in both the basal and the activated (de-repressed) levels of transcription.

Attenuator: This site is termed the attenuator and it lies towards the end of the transcribed leader sequence of 162 nt that precedes the trpE initiator codon.

Structure: The attenuator is a -independent terminator site which has a short GC-rich palindrome followed by eight successive U residues in RNA sequence.

Function: If this sequence is able to form a 3 － 4 hairpin structure in the RNA transcript, then it acts as a highly efficient transcription terminator and only a 140 nt transcript is synthesized.


S3 S4

AA • U

U GC A

C • G G • C C • G C • G C • G G • C A • U

……NNNN UUUUUUU-OH

Rho-independent T.



AttenuationReason of attenuation: Attenuation effect depends on the fact that

transcription and translation are tightly coupled in E. coli; translation can occur as an mRNA is being transcribed.

Sequence 1: The 3’OH-end of the trp leader peptide coding sequence overlaps complementary sequence 1; the two trp codons are within sequence 1.

Stop codon: The stop codon in the leader sequence is between sequence 1 and sequence 2.

3:4 hairpin: It is a conditional terminator (attenuator). When the tryptophan is non-starved, the 3:4 hairpin forms in the mRNA.

Coordination: As transcription of the trp operon proceeds, the RNA polymerase pauses at the end of sequence 2 until a ribosome begins to translate the leader peptide.


Importance of attenuation

700-fold regulatory effect: • Attenuation: The presence of tryptophan gives rise to a 10-fold

repression of trp operon transcription through the process of attenuation alone.

• Trp repressor: Combined with control by the trp repressor (70-fold), this means that tryptophan levels exert a 700-fold regulatory effect on expression from the trp operon.

His operon: For example• Histidine codons: The His operon has a leader sequence which

encodes a peptide with seven successive histidine codons. • Only mechanism: The His operon has no repressor-operator

regulation, and attenuation forms the only mechanism of feedback control.

Other operons: Attenuation occurs in at least six operons that encode enzymes concerned with amino acid bio-synthesis.


L3 Trans. regulation by alternative factor

• Sigma factor

• Heat shock

• Bateriophage factor

hsp47

groel


Regulation patterns and factorsTranscription regulation patterns: • By transcriptional repressors: such as the lac repressor;• By transcriptional activators: such as the CRP;• By different to direct RNApol binding different promoter: Functions of factors : The ’ core enzyme of RNA

polymerase is unable to start transcription at promoter sites. In order to specifically recognize the consensus -35 and -10

elements of the promoters, it requires the factor subunit. This subunit is only required for transcription initiation, being released from the core enzyme after initiation and before

RNA elongation takes place. Features: Many bacteria, including E.coli, produce a set of

factors that recognize different sets of promoters.


Transcriptional regulation by alternative factor in E. coli for stress condition (an example for the use of different )

----- When ＞ 37 ; (42 : very soon; 50 : the only ℃ ℃ ℃products)

Standard 70 ----TTGACA----16-18---------TATAAT------Responsive Promoter -35 -10

Heat shock 32 ----TTGAA-----13-15---CCCCAT-T----------

----- When 37 ; ℃ genes expressed in E.coli by RNApol with 70

More then 17 heat-shock proteins are expressed in E.coli through transcription by RNApol using an alternative 32 , which have own specific promoter consensus sequence

Heat shock gene

T4 in E.coli

Early gene middle genes late genes

host

phage 28 phage -late

Bateriophage factorSome phages provide new subunits to the host RNA polymerase

with a different promoter specificity and hence to selectively express their own phage genes.

This strategy is an effective alternative to the need for the phage to encode its own complete polymerase. This pattern allows its own genes to be transcribed at specific stages during virus infection.

proteins


That’s all for Section L

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Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences Section L Regulation of Transcription in Prokaryotes L1 The lac operon L2