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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 14 GENE REGULATION IN BACTERIA AND BACTERIOPHAGES.

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Presentation on theme: "Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 14 GENE REGULATION IN BACTERIA AND BACTERIOPHAGES."— Presentation transcript:

1 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 14 GENE REGULATION IN BACTERIA AND BACTERIOPHAGES

2 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

3 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

4 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

5 Regulatory proteins have two binding sites One for a small effector molecule The other for DNA

6

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

8 Diauxic Growth Curve Demonstrated Adaptation to Lac Metabolism

9 14-13 Figure 14.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac Operon

10 Regulatory Sequences of the Lac Operon

11 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

12 14-15 Figure 14.4 Constitutive expression RNA pol cannot initiate transcription The lac operon is now repressed

13 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 14-16 Figure 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 Translation The lac operon is now induced

15 The cycle of lac operon induction & repression Figure 14.5 Repressor does not completely inhibit transcription small amounts of the enzymes are made

16 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

17 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

18 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

19 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

20 14-23 Figure 14.7 PaJoMo Experiment

21 Figure

22 Figure

23 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

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

25 Analysis of Lac Operon Mutants - F ’ I - O + Z+Y + I+O+Z-Y+I+O+Z-Y+ lacI

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

27 Analysis of Lac Operon Mutants - - Mutation is cis In merodiploid, LacZ constitutive, but LacY inducible O C only controls transcription of DNA on which O C is located O (operator) is cis-regulatory element

28 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

29 Genetic Implications of Trans vs Cis mutations in trans-acting factors complemented by 2 nd wt gene mutations in cis-acting elements ARE NOT complemented by 2 nd wt element Trans interactions (complementation) indicate mutation in structural gene Cis interactions indicate mutations in regulatory sequences

30 From Jacob & Monod, 1961, J Mol Biol 3:318 Wildtype Induction suppression mutant – Dominant Negative

31 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

32 Analysis of Lac Operon Mutants Mutation is trans Dominant- negative Mutation disrupts ligand binding domain of repressor

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

34 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

35 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

36 Figure 14.8 States of Lac Regulation (b) Lactose but no cAMP

37 Figure 14.8 States of Lac Regulation

38 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 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

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

40 14-47 Organization of the trp operon & regulation via the trp repressor protein Figure Another mechanism of regulation Med

41 14-45 Organization of the trp operon & regulation via the trp repressor protein Figure Cannot bind to the operator site RNA pol can bind to the promoter

42 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

43 Sequence of the trpL mRNA produced during attenuation Figure 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

44 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

45 Organization of the trp operon & regulation via the trp repressor protein Figure Repression occurs

46 Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure Sufficient amounts of tRNA trp 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

47 Possible stem-loop structures formed from trpL mRNA under different conditions of translation Figure Insufficient amounts of tRNA trp Region 1 is blocked 3-4 stem-loop does not form RNA pol transcribes rest of operon Transcription occurs

48 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


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