Regulation of Gene Expression in Bacteria

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Presentation transcript:

Regulation of Gene Expression in Bacteria

Introduction gene regulation Constitutive expression the amount of gene expression can vary under different conditions Constitutive expression constant amounts of expression sometimes called “housekeeping genes” Ubiquitous expression Expressed in all cell types (multicellular organisms) Differential expression Temporal & spatial expression regulated genes expressed only when required

Transcriptional Regulation Most regulation 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 increase transcription inducer Bind repressors & prevent repressor from binding DNA co-activator Bind activators & cause activator to bind DNA Genes regulated this way are inducible decrease transcription co-repressor bind repressors & cause repressor to bind DNA inhibitor bind activators & prevent activator from binding DNA Genes regulated this way are repressible

Inducible Expression Co-activator

Repressible Control

gene regulation gods François Jacob & André Lwoff – 1953 CSH Symposium Jacques Monod – Paris 1961 François Jacob & André Lwoff – 1953 CSH Symposium

Diauxic Growth Curve Demonstrated Adaptation to Lac Metabolism

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

Regulatory Sequences of the Lac Operon Stop 1 10 Operator 3

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 Activation cAMP binds CAP protein, cAMP-CAP dimerizes & binds CAP site in DNA Insures operon is on ONLY if lactose is present glucose is low

RNA pol cannot initiate transcription Constitutive expression 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.

The lac operon is now induced Translation The lac operon is now induced The conformation of the repressor is now altered Repressor can no longer bind to operator Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The cycle of lac operon induction & repression Repressor does not completely inhibit transcription So very small amounts of the enzymes are made 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 Introduced into mutant bacteria by conjugation Bacteria that receive 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’ plasmid 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

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

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 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 Dominant-negative Mutation disrupts ligand binding domain of repressor

Analysis of Lac Operon Mutants Mutation disrupts DNA binding domain of repressor

3 categories of dominant mutations Figure 14.9

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 (b) Lactose but no cAMP

States of Lac Regulation Figure 14.8

The trp Operon The trp operon encodes enzymes for biosynthesis of tryptophan trpE, trpD, trpC, trpB & trpA encode enzymes trpR & trpL involved in regulation trpR  Encodes trp repressor protein Functions in repression trpL  Encodes a short peptide called the Leader peptide Functions in attenuation

RNA pol can bind to the promoter Cannot bind to the operator site Organization of the trp operon & regulation via the trp repressor protein Figure 14.13

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

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

Attenuation occurs in bacteria because of the coupling of transcription & translation During attenuation, transcription begins but it is terminated before 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 Thus attenuation inhibits the further production of tryptophan The trpL segment of trp operon immediately downstream from the operator site plays a critical role in attenuation It encodes a short peptide termed the Leader peptide

Region 2 is complementary to regions 1 & 3 Multiple stem-loops structures are possible The 3-4 stem loop is followed by a sequence of Uracils It acts as an intrinsic (r-independent) terminator These two codons provide a way to sense if there is sufficient tryptophan for translation Sequence of the trpL mRNA produced during attenuation

Structure of trpL 41

There are three possible scenarios 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. No translation 2. Low levels of tryptophan 3. High levels of tryptophan Refer to Figure 14.15 14-50 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Transcription terminates just past the trpL gene Most stable form of mRNA occurs Therefore no coupling of transcription & translation Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure 14.15 14-51

44

Sufficient amounts of tRNAtrp 3-4 stem-loop forms Translation of the trpL mRNA progresses until stop codon Transcription terminates RNA polymerase pauses Region 2 cannot base pair with any other region Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure 14.15 14-53

46

Region 1 is blocked 3-4 stem-loop does not form RNA pol transcribes rest of operon Insufficient amounts of tRNAtrp Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure 14.15 14-52

48

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 14-54 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

PaJoMo Experiment

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