molecular biology lecture 7

8/12/2019 Molecular Biology Lecture 7

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BIOL321 - Prokaryote gene regulation

Madigan et al. 2003. Biology of Microorganisms. Prentice Hall



Objectives

- Overview of Prokaryotic gene transcription

- Introduction of gene regulation

- Promoters

- Operators

- Introduction to lac regulation

- History

- Lactose breakdown

- Induction of lac operon

- Mutations of lac

- The hunger signal and cAMP signaling- Introduction to t rp regulation

- Roles and functions of trp

- Repression of trp by TrpR

- Translational control of trp through attenuation



A Quick Review of Transcription

• The step where a molecule of mRNA is

assembled, based on DNA

• RNA polymerase is responsible for

reading the DNA and assembling the

growing mRNA strand



Prokaryotic vs. Eukaryotic

Transcription



Getting Started

• RNA polymerase must find a promoter

sequence in DNA before it can start

transcribing

• The polymerase can bind the DNA

directly (in bacteria) or seek outtranscription factors that bind to the

promoter (in eukaryotes)



In bacteria…

DNA

Promoter

RNA polymerase

Transcription Start

Site

RNA Polymerase (RNAP) ‘scans’ along the DNA,

looking for a promoter



In bacteria…

DNA

Promoter

Transcription Start

Site

When a promoter sequence is recognized, RNAP ‘melts’ the D



In bacteria…

DNA

Promoter

Transcription Start

Site

RNAP starts synthesizing the mRNA at the transcription start si



Transcription: Always On?

• Some genes (constitutive genes) are always

expressed within a cell

• However, other genes only need to beexpressed at certain times – inducible genes

• Therefore, to reduce wasted effort, many

genes are regulated , and only expressed

under certain conditions



Regulating Your Genes

• This regulation can occur at many

different steps of gene expression,

including: – Transcription

– mRNA processing (several kinds)

– Translation

• However, the majority of regulation

takes place at the transcriptional level



Regulating Your Genes• In bacteria, regulated genes have an

downstream region adjacent to the promotercalled the operator

• The operator is a binding site for proteins thathelp to regulate gene expression

DNA

Promoter Operator



Regulatory Proteins

• There are two main types of regulatory proteins in

bacteria:

– Repressors bind to the operator and prevent RNA

polymerase from initiating transcription – Activators bind to sequences near the promoter

and allow RNA polymerase to initiate transcription

• A given gene (or group of genes) may use either or

both types of protein for regulation



Regulatory Proteins

DNA

Promoter Operator

Repressor – Transcription Blocked

Activator – Transcription EnabledDNA

Promoter Operator

RNAP

repressor

activator



The lac Operon



The lac operon

• Jacob and Monod studied the regulation ofgenes required for the metabolism of lactose

in bacteria (coordinate regulation)

François Jacob

(1920 - )

Jacques Monod

(1910 – 1976)

Won the Nobel Prize in Physiology or Medicine, 1965



Lactose Catabolism

• A transcriptionally regulated system

• When the bacterium is in an environment that contains lactose,

the cell would want to turn on genes for enzymes that are

required for lactose catabolism• When lactose is absent, the cell would want to turn these genes

OFF

• Actual regulation is more complicated

• Glucose = prime source of food

• So, if both glucose and lactose are available, the bacterium willturn off lactose metabolism in favour of glucose



Lactose Breakdown in E. co li

by the enzyme beta-galactosidase

+

GLUCOSE GALACTOSE



The lac operon

• Studied extensively by François Jacob and Jacques Monod• If lactose is present then three protein products are made:

– Lac Z = -galactosidase:

(lactose glucose +galactose)(lactose allolactose)

– Lac Y = Permase

(active transport of lactose across cell membrane)

– Lac A = Transacetylase

(galactose acetylgalactose)



The lac operon

• All three genes share the same promoter (lacP ), the same

operator (lacO), and are transcribed as a single mRNA

( polycistronic mRNA)

• The gene for a regulatory protein (LacI), encoding a

repressor, is found near the operon

lacI lacP lacO lacZ lacY lacA

lac operon



lac Operon Regulation

• Since the role of proteins encoded by lac isto break down the sugar lactose, the

structural (protein-coding) genes lacZ, lacYand lacA should only be expressed whenlactose is present in the cell

•

The lacI gene encodes a repressor proteinthat shuts the system down when lactose isnot present



Induction of the lac operon

• If lactose is present in the environment (growth medium), the lac

operon is ‘induced’

• Lactose enters the cell and binds to the lac operon

• This induces a conformational change that allows the repressor to

fall off the DNA

– Now RNA polymerase is free to move along the DNA

– RNA can be made from the three genes

– Lactose can be metabolized

• When the inducer (lactose) is removed, the repressor returns to its

original conformation and binds to the DNA

– RNA polymerase can no longer get past the promoter

– No RNA or protein is made



Induction of the lac operon -1

Repressor (LacI) bound to operator, RNA

polymerase cannot initiate transcription

at the promoter

No lactose - system turned OFF

lacP lacO lacZ lacY lacA

LacI




If lactose is added, an isomer (allolactose) will bind

to the repressor


Allolactose




The allolactose-bound repressor undergoes a conformational

change and dissociates from the operator sequence.

RNA polymerase is then free to initiate transcription

Lactose present – system turned ON




Mutations affecting lac

• lacZ -, lacY -, lacA- Structural gene mutations lead to

non-functional proteins

• lacP - Non-functional promoter, RNAP cannot bind so

genes will not be expressed• lacOC Non-functional operator so repressor cannot

bind. Since the system cannot be shut off, this is a

constitutive mutation




Mutations affecting the

lac operon

• lacI - Non-functional repressor, unable to

bind the operator to shut off transcription

• lacI S Super-repressor, unable to dissociatefrom operator. System always off.




Summary of the E.coli lactose operon



Regulation of lac

• When a repressor is used to turn the system off, that

system is under negative control . The LacI repressor

is an example of this.

• The lac operon is also under positive control , where

an activator protein is used to increase the efficiency

of transcription



Glucose is the Favourite

Carbon Source of E. co li • Whereas E. coli can metabolize lactose using the

products of the lac operon, it actually ‘prefers’ glucose

as its carbon source• Remember that glucose can be directly introduced into

glycolysis

• Therefore, when glucose is present, there is no need to

express the genes in the lac operon



No glucose?

• If a cell runs out of glucose, a small molecule – cyclic3’5’adenosine monophosphate (cAMP) – is producedfrom ATP by the enzyme adenylate cyclase

• cAMP is a ‘hunger signal’ that stimulates the

expression of genes that produce enzymes to breakdown alternate sugars, such as lactose

• cAMP binds the activator protein CRP (cAMPreceptor protein) or CAP (catabolite activatorprotein), which can then bind to lacP to help activatetranscription



The Hunger Signal


CRP binding site

Inactive CRP

When glucose is present (NOT hungry), no

cAMP is produced so CRP is inactive. RNAP

cannot initiate transcription of the lac operon.

RNAP



The Hunger Signal

If glucose is depleted, cAMP is produced, which

binds to and activates CRP. Activated CRP can

then bind to the promoter.


CRP binding site

Active CRPcAMP



The Hunger Signal

2 Actions combine:• Lactose present – then allolactose will bind to repressor to cause its

dissociation.

• Activated CRP binds the promoter, so RNAP can enter and initiate

transcription

• Genes of the lac operon are transcribed


allolactose bound to repressor



On vs. Off

Lactose

present

Lactose

absent

Glucose present Glucose absent

No repressor bound

No activator bound

OFF

Repressor bound

No activator bound

OFF

No repressor bound

Activator bound

ON

Repressor bound

Activator bound

OFF

Note: this option would occur

when a third kind of sugar was present



The t rp Operon



• In addition to sugars, like glucose and lactose, E. coli cells

also require amino acids

• One essential amino acid is tryptophan

• When E. coli is in tryptophan (milk and poultry) it will absorb

the amino acids from the media

• When tryptophan is not present in the media then the cell

must manufacture its own amino acids

The t rp Operon



The t rp Operon

• Responsible for biosynthesis of the essential amino acidtryptophan

• If tryptophan levels are low, turn on expression toproduce more

• If tryptophan levels are high, turn off the system

• All 5 genes are transcribed together as a unit

trpP trpO trpE trpD trpC trpB trpA



The t rp Repressor (TrpR)

• Encoded by the trpR gene, located far from the trp

operon

• TrpR can only bind to the trp operator if activated by

a tryptophan molecule (co-repressor)

trpP trpO trpE trpD trpC trpB trpA

TrpR bound to tryptophan



Translational Control of t rp

gene expression

• Another method of control is through attenuation • The first protein-coding gene in the trp operon is the‘leader peptide’ trpL, which contains adjacent codonsfor tryptophan

• Therefore, if tryptophan - tRNA is abundant in the

cell, translation of this peptide will be quick



Attenuation

• mRNA secondary structure is essential in thisregulation

• If tryptophan is abundant, the ribosome willspeed quickly through the trp codons

BUT

The mRNA BEHIND the codons will form astem-loop terminator structure, and RNAP willfall off before it can transcribe trpEDCBA(slide to come)



Attenuation

• If tryptophan is rare in the cell, the ribosome

will pause at the Trp codons

• This pausing will prevent the formation of theterminator signal, and RNAP will continue

transcribing the trpEDCBA genes

Stem loops form by complimentary base pairing



Stem loops form by complimentary base pairing

in the Trp leader RNA

Movement of the ribosome through the Trp codons produces alternate stem

loops:

Fast movement (Trp present) = termination structure (UUUUU) (no

transcription)

2 3

Tryptophan



trpE trpD trpC trpB trpATrp

Codons Ribo

4

Stem loop

Termination

structure

RNA

pol.

RNA polymerase

falls offRibosome

proceeds quickly

through Trp

codons

trpE trpD trpC trpB trpATrp

Codons

2 3

4

AttenuationRibosome stalls

at Trp codons

Transcription

proceeds through

Trp Operon

yp p

high

Tryptophan

low

RNA

pol.

Ribo

molecular biology lecture 7

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