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CHAPTER 14

GENE REGULATION IN BACTERIA AND BACTERIOPHAGES

Introduction

The term gene regulation means that the level of gene expression can vary under different conditions

Genes that have constant levels of expression are termed constitutive sometimes called “housekeeping genes”

The benefit of regulating genes is that encoded proteins will be produced only when required

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

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

Negative control refers to transcriptional regulation by repressor proteins

Positive control to regulation by activator proteins

Transcriptional Regulation

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

Regulatory proteins have two binding sites

One for a small effector molecule

The other for DNA

gene regulation gods

François Jacob & André Lwoff – 1953 CSH SymposiumJacques Monod – Paris 1961

Diauxic Growth Curve Demonstrated Adaptation to Lac Metabolism

14-13Figure 14.3

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The lac Operon

Regulatory Sequences of the Lac Operon

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 site

in DNA

Insures that operon is on only if lactose is present glucose is low

The Lac Operon Is Regulated both Positively & Negatively

14-15Figure 14.4

Constitutive expression

RNA pol cannot initiate transcription

The lac operon is now repressed

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 lac operon 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.

14-16Figure 14.4

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The conformation of the repressor is now altered

Repressor can no longer bind to operator

TranslationThe lac operon is now induced

The cycle of lac operon induction & repressionFigure 14.5

Repressor does not completely inhibit transcription

small amounts of the enzymes are made

1950s, Jacob & Monod, & Arthur Pardee, identified mutant bacteria with abnormal lactose adaptation

defect in lacI gene designated lacI– I = induction mutant caused constitutive expression of lac operon

(ie in absence of lactose)

The lacI– mutations mapped very close to the lac operon

The lacI Gene Encodes a Repressor Protein

Jacob, Monod & Pardee hypothesized 2 ways for lacI to function

Used genetic approach to test hypotheses

This hypothesis predicts that lacI works in trans manner

This hypothesis predicts that lacI works in a cis manner

Used F’ plasmids carrying part of lac operon Put into mutant bacteria by conjugation Bacteria that get F’ have 2 copies of lacI

gene merodipoloids

PaJaMo Experiment

2 lacI genes in a merodiploid are alleles lacI– on the chromosome lacI+ on the F’ factor

Genes on F’ plasmid are trans to bacterial chromosome 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 lac operon on the

bacterial chromosome, because they are not physically adjacent

PaJaMo Experiment

14-23Figure 14.7

PaJoMo Experiment

Figure 14.7 14-24

Figure 14.7 14-25

Results

Lactose addition has no effect because operon is already on Induction is restored in merodiploid.

Now lactose addition is required to turn operon on

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

Wildtype

Induction mutants

Analysis of Lac Operon Mutants

-

F’I-O+Z+Y+

I+O+Z-Y+

lacI

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

Analysis of Lac Operon Mutants

-

-

Mutation is cis

• In merodiploid, LacZ constitutive, but LacY inducible

• OC only controls transcription of DNA on which OC is located

• O (operator) is cis-regulatory element

Interpreting the Data

The interaction between regulatory proteins & DNA sequences have led to two definitions Trans-effect & trans-acting factor

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 lac repressor on the lac operon

Cis-effect & cis-acting element A DNA sequence adjacent to the gene(s) it regulates Mediated by sequences that are bound by regulatory proteins Example: The lac operator

Genetic Implications of Trans vs Cis

mutations in trans-acting factors complemented by 2nd wt gene

mutations in cis-acting elements ARE NOT complemented by 2nd wt element

Trans interactions (complementation) indicate mutation in structural gene

Cis interactions indicate mutations in regulatory sequences

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

Wildtype

Induction suppression mutant – Dominant Negative

Dominant Inhibitors or Dominant 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

Analysis of Lac Operon Mutants

Mutation is trans

Dominant-negative

Mutation disrupts ligand binding domain of repressor

Analysis of Lac Operon Mutants

Mutation disrupts DNA binding domain of repressor

catabolite repression

When exposed to both lactose & glucose E. coli uses glucose first, & catabolite repression

prevents the use of lactose When glucose is depleted, catabolite repression is

alleviated, & the lac operon is expressed

The sequential use of two sugars by a bacterium is termed diauxic growth

lac Operon Also Regulated By Activator Protein

Effector molecule in catabolite repression cAMP (cyclic AMP)

cAMP is produced from ATP by adenylyl cyclase

cAMP binds activator protein CAP or CRP (Catabolite Activator Protein) or (cyclic AMP receptor protein)

The lac Operon Is Also Regulated By an Activator Protein

Figure 14.8

States of Lac Regulation

(b) Lactose but no cAMP

Figure 14.8

States of Lac Regulation

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The trp operon (pronounced “trip”) is involved in the biosynthesis of the amino acid tryptophan

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

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

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

Functions in attenuation

The trp Operon

14-44

Organization of the trp operon & regulation via the trp repressor protein

Figure 14.13

14-47

Organization of the trp operon & regulation via the trp repressor protein

Figure 14.13

Another mechanism of regulation

Med

14-45

Organization of the trp operon & regulation via the trp repressor protein

Figure 14.13

Cannot bind to the operator site

RNA pol can bind to the promoter

Attenuation occurs in bacteria because of the coupling of transcription & translation

During attenuation, transcription actually begins but it is terminated 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 of trp 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

Sequence of the trpL mRNA produced during attenuationFigure 14.14

These two codons provide a way to sense if there is sufficient

tryptophan for translation

The 3-4 stem loop is followed by a sequence

of Uracils

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

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

Organization of the trp operon & regulation via the trp repressor protein

Figure 14.13

Repression occurs

Possible stem-loop structures formed from trpL mRNA under different conditions of translation

Figure 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 terminates

RNA polymerase pauses

Med

Attenuation occurs

Possible stem-loop structures formed from trpL mRNA under different conditions of translation

Figure 14.15

Insufficient amounts of tRNAtrp

Region 1 is blocked

3-4 stem-loop does not form

RNA pol transcribes rest of operon

Transcription occurs

The study of many operons revealed a general trend concerning 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 a substance) are typically repressible

The inhibitor or corepressor is the small molecule that is the product of the operon

Inducible vs Repressible Regulation