CHAPTER 14

GENE REGULATION IN BACTERIA AND 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 are termed constitutive

sometimes called housekeeping genes

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

Transcriptional Regulation

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 bind activators & prevent activator from binding DNA

Inhibitors

Genes regulated this way are repressible

Regulatory proteins have two binding sites

One for a small effector molecule The other for DNA

gene regulation gods

Jacques Monod Paris 1961

Franois Jacob & Andr Lwoff 1953 CSH Symposium

Diauxic Growth Curve Demonstrated Adaptation to Lac Metabolism

The lac Operon

Figure 14.3

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Regulatory Sequences of the Lac Operon

The Lac Operon Is Regulated both Positively & Negatively

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 cAMP binds CAP protein, cAMP-CAP dimerizes & binds CAP site in DNA

Activation

Insures that operon is on only if

lactose is present glucose is low

RNA pol cannot initiate transcription

Constitutive expression

The lac operon is now repressed

Figure 14.4

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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 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.

The lac operon is now induced

Translation

The conformation of the repressor is now altered Repressor can no longer bind to operator

Figure 14.4

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Repressor does not completely inhibit transcription small amounts of the enzymes are made

Figure 14.5

The cycle of lac operon induction & repression

The lacI Gene Encodes a Repressor Protein

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

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

This hypothesis predicts that lacI works in trans manner

This hypothesis predicts that lacI works in a cis manner

Used genetic approach to test hypotheses

PaJaMo Experiment

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 binding site on F plasmid cannot affect lac operon on the bacterial chromosome, because they are not physically adjacent

If hypothesis 2 is correct

PaJoMo Experiment

Figure 14.7

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Figure 14.7

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Figure 14.7

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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

Wildtype Induction mutants

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

Analysis of Lac Operon Mutants

-

lacI

I+O+Z-Y+ FI-O+Z+Y+

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

Wildtype

Induction suppression mutant Dominant Negative

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

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 Dominantnegative

Mutation disrupts ligand binding domain of repressor

Analysis of Lac Operon Mutants

Mutation disrupts DNA binding domain of repressor

lac Operon Also Regulated By Activator Protein

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

The lac Operon Is Also Regulated By an 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)

States of Lac Regulation

Figure 14.8

(b) Lactose but no cAMP

States of Lac Regulation

Figure 14.8

The trp Operon

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 Functions in attenuation

trpL Encodes a short peptide called the Leader peptide

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Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein

Med

Another mechanism of regulation

Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein

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RNA pol can bind to the promoter

Cannot bind to the operator site

Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein

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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

Region 2 is complementary to regions 1 & 3 Region 3 is complementary to regions 2 & 4 Therefore several stem-loops structures are possible
The 3-4 stem loop is followed by a sequence of Uracils It acts as an intrinsic (-independent) terminator

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

Figure 14.14 Sequence of the trpL mRNA produced during attenuation

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

Figure 14.13 Organization of the trp operon & regulation via the trp
repressor protein

Attenuation occurs
Sufficient amounts of tRNAtrp Translation of the trpL mRNA progresses until stop codon RNA polymerase pauses Region 2 cannot base pair with any other region Transcription terminates 3-4 stem-loop forms

Med

Figure 14.15 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation

Transcription occurs
Region 1 is blocked 3-4 stem-loop does not form

RNA pol transcribes rest of operon

Insufficient amounts of tRNAtrp

Figure 14.15 Possible stem-loop structures formed from trpL mRNA under
different conditions of translation

Inducible vs Repressible Regulation

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