Prok Gene Reg

7/31/2019 Prok Gene Reg

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Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

CHAPTER 14

GENE REGULATION IN BACTERIAAND BACTERIOPHAGES


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Introduction

The term gene regulation means that the level of

gene expression can vary under different conditions

Genes that have constant levels of expression aretermed constitutive sometimes called housekeeping genes

The benefit of regulating genes is that encoded

proteins will be produced only when required


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Most regulation of gene expression is attranscriptional level rate of RNA synthesis increased or decreased

Transcriptional regulation involves actions of twotypes of regulatory proteins Repressors Bind to DNA & inhibit transcription Activators Bind to DNA & increase transcription

Negative control refers to transcriptional regulationby repressor proteins

Positive control to regulation by activator proteins

Transcriptional Regulation


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Small effector molecules affect transcription regulation bind to regulatory proteins not to DNA directly

effector molecule may increase transcription inducers

Bind activators & cause activator to bind DNA

Bind repressors & prevent repressor from binding DNA Genes regulated this way are inducible

effector molecule may inhibit transcription Corepressors

bind repressors & cause repressor to bind DNA

Inhibitors bind activators & prevent activator from binding DNA

Genes regulated this way are repressible

Transcriptional Regulation


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Regulatory proteins havetwo binding sites One for a small effector

molecule The other for DNA


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gene regulation gods

Franois Jacob & Andr Lwoff 1953 CSH SymposiumJacques Monod Paris 1961


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Diauxic Growth Curve DemonstratedAdaptation to Lac Metabolism


9/4814-13Figure 14.3 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lacOperon


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Regulatory Sequences of the Lac Operon


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Negative - repressor protein - LacI

Positive - activator protein CAP or CRP

Induction of Lac operon requires 2 events

Release of repression lactose binds to the lac repressor causing the repressor to release

operator site in DNA

Activation

cAMP binds CAP protein, cAMP-CAP dimerizes & binds CAP sitein DNA

Insures that operon is on only if lactose is present

glucose is low

The LacOperon Is Regulated both

Positively & Negatively


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Constitutive

expression

RNA pol

cannot initiate

transcription

The lacoperon is now

repressed


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Lac repressor protein (violet)forms a tetramer which binds to

two operator sites (red) located

93 bp apart in the DNA causing

a loop to form in the DNA. As a

result expression of the lacoperon is turned off. This model

also shows the CAP protein

(dark blue) binding to the CAP

site in the promoter (dark blue

DNA). The -10 & -35 sequences

of the promoter are indicated in

green.


14/4814-16Figure 14.4 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The conformation of the

repressor is now altered

Repressor can no longer

bind to operator

TranslationThe lacoperon is now

induced


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The cycle oflacoperon induction & repressionFigure 14.5

Repressor does not completely

inhibit transcription

small amounts of the enzymes

are made


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1950s, Jacob & Monod, & Arthur Pardee, identified

mutant bacteria with abnormal lactose adaptation

defect in lacIgene designated lacII = induction mutant

caused constitutive expression oflacoperon

(ie in absence of lactose)

The lacI mutations mapped very close to the lacoperon

The lacIGene Encodes a Repressor

Protein


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Jacob, Monod & Pardee hypothesized 2 ways forlacI

to function

Used genetic approach to test hypotheses

This hypothesis predicts that

lacI works in trans manner This hypothesis predicts thatlacI works in a cis manner


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Used F plasmids carrying part oflacoperon Put into mutant bacteria by conjugation

Bacteria that get F have 2 copies oflacI

gene merodipoloids

PaJaMo Experiment


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2 lacIgenes in a merodiploid are alleles lacI on the chromosome lacI+ on the F factor

Genes on F plasmid are trans to bacterialchromosome If hypothesis 1 is correct

repressor produced from F plasmid can regulate the lac

operon on the bacterial chromosome If hypothesis 2 is correct

binding site on F plasmid cannot affect lacoperon on thebacterial chromosome, because they are not physically

adjacent

PaJaMo Experiment


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14-23Figure 14.7

PaJoMo Experiment


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Figure 14.7 14-24


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Figure 14.7 14-25


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Results

Lactose addition has no effect

because operon is already onInduction is restored in merodiploid.

Now lactose addition is required to

turn operon on


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From Jacob & Monod, 1961,J Mol Biol3:318

Wildtype

Induction

mutants

Analysis of Lac Operon Mutants


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-

FI-O+Z+Y+

I+O+Z-Y+

lacI

From Jacob & Monod, 1961, J Mol Biol 3:318


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l f O


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-

-

Mutation is

cis

In merodiploid, LacZ constitutive, but LacY inducible

OC

controls transcription of DNA on which OC

islocated

O (operator) is cis-regulatory element


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Interpreting the Data

The interaction between regulatory proteins & DNAsequences have led to two definitions Trans-effect & trans-actingfactor

Genetic regulation that can occur even though DNA segments are

not physically adjacent Mediated by genes that encode DNA-binding regulatory proteins

Example: The action of the lacrepressor on the lacoperon

Cis-effect & cis-actingelement A DNA sequence adjacent to the gene(s) it regulates

Mediated by sequences that are bound by regulatory proteins

Example: The lacoperator


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Genetic Implications ofTrans vs Cis

mutations in trans-acting factors complemented by2nd wt gene

mutations in cis-acting elements ARE NOTcomplemented by 2nd wt element

Trans interactions (complementation) indicate

mutation in structural gene

Cis interactions indicate mutations in regulatory

sequences


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Wildtype

Induction

suppression

mutant

Dominant

Negative


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Dominant Inhibitors orDominant Negatives

Proteins with multiple functional domains &form multimeric complexes may be altered

to prevent one function, but allow the other When mutants retain ability to form

multimeric complexes, dominant inhibition

may occur



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Mutation is

trans

Dominant-negative

Mutation disrupts ligand binding domain of repressor



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Mutation disrupts DNA binding domain of repressor


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catabolite repression

When exposed to both lactose & glucose E. coliuses glucose first, & catabolite repression prevents

the use of lactose

When glucose is depleted, catabolite repression is

alleviated, & the lacoperon is expressed

The sequential use of two sugars by a bacterium is

termed diauxic growth

lacOperon Also Regulated By Activator Protein

The lac Operon Is Also Regulated By an Activator


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Effector molecule in catabolite repression cAMP

(cyclic AMP)

cAMP is produced from ATP by adenylyl cyclase

cAMP binds activator protein CAP orCRP(CataboliteActivator Protein) or(cyclic AMP receptor protein)

The lacOperon Is Also Regulated By an Activator

Protein

States of Lac Regulation


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Figure 14.8


(b) Lactose but no cAMP

States

of Lac Regulation


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Figure 14.8



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Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The trp operon (pronounced trip) is involved in the

biosynthesis of the amino acid tryptophan

The genes trpE, trpD,trpC, trpB & trpA encode enzymesinvolved in tryptophan biosynthesis

The genes trpR& trpL are involved in regulation trpREncodes the trp repressor protein

Functions in repression

trpL Encodes a short peptide called the Leader peptide Functions in attenuation

The trp Operon

14-44


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Organization of the trp operon & regulation via the trp

repressor protein

Figure 14.13


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14-47


repressor protein

Figure 14.13

Another mechanism

of regulation

Med


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14-45


repressor protein

Figure 14.13

Cannot bind to

the operator site

RNA pol can bind

to the promoter


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Attenuation occurs in bacteria because of the coupling of

transcription & translation

During attenuation, transcription actually begins but it isterminated before the entire mRNA is made A segment of DNA, termed the attenuator, is important in facilitating

this termination

In the case of the trp operon, transcription terminates shortly past

the trpL region (Figure 14.13c) Thus attenuation inhibits the further production of tryptophan

The segment oftrp operon immediately downstream from the

operator site plays a critical role in attenuation The first gene in the trp operon is trpL

It encodes a short peptide termed the Leader peptide

R i 2 i l i 1 & 3


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Sequence of the trpL mRNA produced during attenuationFigure 14.14

These two codons provide a wayto sense if there is sufficient

tryptophan for translation

The 3-4 stem loop is

followed by a sequence ofUracils

Region 2 is complementary to regions 1 & 3

Region 3 is complementary to regions 2 & 4

Therefore several stem-loops structures are possible

It acts as an intrinsic

( -independent) terminator


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Therefore, the formation of the 3-4 stem-loop

causes RNA pol to terminate transcription at the

end of the trpL gene

Conditions that favor the formation of the 3-4

stem-loop rely on the translation of the trpL mRNA

There are three possible scenarios 1. High levels of tryptophan

2. Medium levels of tryptophan high trp-tRNA

3. Low levels of tryptophan med-low trp-tRNA

Repression occurs


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repressor protein

Figure 14.13

Repression occurs

Attenuation occurs


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Possible stem-loop structures formed from trpL mRNA under

different conditions of translationFigure 14.15

Sufficient amounts of tRNAtrp

Translation of the trpL mRNA

progresses until stop codon

Region 2 cannot base pair

with any other region

3-4 stem-loop forms

Transcription

terminatesRNA polymerase pauses

Med

Attenuation occurs

ranscr p on occurs


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Possible stem-loop structures formed from trpL mRNA under

different conditions of translationFigure 14.15

Insufficient amounts

of tRNAtrp

Region 1 is blocked

3-4 stem-loop

does not form

RNA pol transcribes

rest of operon

ranscr p on occurs


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The study of many operons revealed a general trendconcerning inducible versus repressible regulation

Operons involved in catabolism (ie. breakdown of a

substance) are typically inducible The substance to be broken down (or a related compound) acts

as the inducer

Operons involved in anabolism (ie. biosynthesis of asubstance) are typically repressible The inhibitor or corepressor is the small molecule that is the

product of the operon

Inducible vs Repressible Regulation

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