1. Chapter 17 Regulation of gene expression in bacteria: lac Operon of E. coli trp operon of E. coli

2. Gene expression in bacteria - some useful distinctions: Regulated genes Control cell growth and cell division. Expression is regulated by the needs of the cell and the environment as needed (not continuously). Constitutive genes Continuously expressed. Housekeeping genes (such as those required for protein synthesis and glucose metabolism). *All genes are regulated at some level!

3. Operon - what is it? Cluster of genes in which expression is regulated by operator-repressor protein interactions, operator region, and the promoter. Contents of an operon: Promoter Repressor Operator (controlling site) Coding sequences Terminator Adjacent polycystronic coding sequences (e.g., bacteria, mtDNA) are co-transcribed to make a polygenic mRNA. Inducers and induction: Inducer = chemical or environmental agent that initiates transcription of an operon. Induction = synthesis of gene product(s) in response to an inducer.

4. Fig. 17.1, Organization of an inducible gene containing an operon.

5. E. coli’s lac operon: E. coli expresses genes for glucose metabolism continuously. Metabolism of other alternative types of sugars (e.g., lactose) are regulated specifically. Lactose = disaccharide (glucose + galactose), provides energy. Lactose acts as an inducer (effector molecule) and stimulates expression of three proteins at 1000-fold increase: -galactosidase (lacZ) Breaks lactose into glucose + galactose. Converts lactose to the allolactose, regulates lac operon. Lactose permease (lacY) Transports lactose across cytoplasmic membrane. Transacetylase (lacA) transfers an acetyl group from acetyl-CoA to -galactosides.

6. Organization of the E. coli lac operon: Francois Jacob and Jacques Monod (Pasteur Institute, Paris, France) Studied the organization and control of the lac operon in E. coli. Earned Nobel Prize in Physiology or Medicine 1965. Studied 2 different types of mutations in the lac operon: 1.Mutations in protein-coding gene sequences. 2.Mutations in regulatory sequences.

7. 1. Mutations in protein-coding gene sequences mapped gene locations: 1.lacZ (-galactosidase) knock-out mutants inhibit function of lactose permease (lacY) and transacetylase (lacA). 2.lacY (lactose permease) knock-out mutants inhibit function of transacetylase (lacA), but do not affect -galactosidase (lacZ). 3.lacA (transacetylase) knock-out mutants do not affect - galactosidase (lacZ) or lactose permease (lacY). Conclusion: 3 lac operon genes are linked in the following order: 1.lacZ codes -galactosidase 2.lacY codes lactose permease 3.lacA codes transacetylase

8. Fig. 17.3, Translation of lac operon in wild type and mutant E. coli.

9. 2. Analysis of mutations in regulatory sequences affecting gene expression: Jacob and Monod also studied mutants that produced lac operon proteins whether or not lactose (inducer) is present: Predicted 2 types of upstream lac regulatory mutants exist: 1.Mutations in the lac operator (lacO) 1.Mutation in the lac repressor (lacI)

10. Mutations in the lac operator (lacO): Used diploid E. coli strains, containing lac operon genes with normal promoters on extra-chromosomal plasmids (F’). F’lacO+lacZ-lacY+ permease (only with lactose) ClacOclacZ+lacY- -galactosidase (lactose absent) (continuously expressed) Conclusions: 1.lacO occurs upstream of lacZ and lacY and affects production of proteins downstream on the same molecule. 2.lacO is a regulatory sequence; no diffusible product is produced. 3.If diffusible product is produced, expect permease production in absence of lactose.

11. Mutations in the lac repressor (lacI): Also used partial diploid E. coli F’ strains, containing lac operon genes with normal promoters and normal operators. F’lac I+lacO+lacZ-lacY+ ClacI-lacO+lacZ+lacY- In absence of lactose, no -galactosidase or permease are produced. In presence of lactose, -galactosidase and permease are synthesized (lactose is an inducer). Conclusion: 1.lacI+ produces a repressor protein (a diffusible product). 2.In absence of lactose, repressor protein binds to the operator and inhibits synthesis of downstream proteins. 3.In presence of lactose, repressor protein is inhibited by allolactose, and downstream protein synthesis occurs.

12. Mutations also occur in the promoter (P lac ): Inhibit RNA polymerase binding and protein synthesis with or without presence of lactose. Single mutation affects all three protein coding genes, lacZ, lacY, and lacA.

13. Fig. 17.4, General organization of the lac operon of wild-type E. coli. Order of controlling elements and genes: lacI:promoter-lacI-terminator operon:promoter-operator-lacZ-lacY-lacA-terminator

14. Fig. 17.5, Functional state of the E. coli lac operon in the absence of lactose:

15. Fig. 17.7, Functional state of the E. coli lac operon growing on lactose:

16. Fig. 17.6, Model of lac repressor tetramer (4 polypetides) protein.

17. Summary of Jacob-Monod E. coli lac operon model: 1.Operon is a cluster of genes; expression is regulated by operator- repressor protein interactions, operator, and a promoter. 2.lac I gene possesses its own weak promoter and terminator; lacI repressor proteins always exist in low concentration. Repressor protein is a tetramer (4 polypeptides). Repressor binds the operator (lacO) and prevents RNA polymerase initiation of transcription. Binding is not complete, so low levels of lacZ, lacY, and lacA proteins are always synthesized. As soon as lactose occurs in high concentration, lac operon switches to the “on” position. 3.-galactosidase in wild-type E. coli growing on lactose converts lactose to allolactose. Allolactose binds to repressor proteins, which in turn are “disabled” and unable to bind the operator. Allolactose induces expression of the lac operon.

18. Summary of Jacob-Monod E. coli lac operon model (cont.): 4.RNA polymerase initiates synthesis of a single polygenic mRNA containing mRNA for lacZ, lacY, and lacA. 5.mRNA is translated as a single molecule by a string of ribosomes. 6.lac operon is said to be under negative control (lacI blocks RNA polymerase if inducer is absent). 7.Different types of mutations occur in lacO, lacI, and promotor: lacO-change repressor binding site (repressor does not bind) -continuously expressed lacI-change repressor conformation (cannot bind operator) -continuously expressed -super-repressors bind operator but not allolactose -lactose does not induce the operon promoter-alter affinity for RNA polymerase -increase or decrease transcription rate

19. Positive control also occurs in the lac operon: Positive control occurs when lactose is E. coli’s sole carbon source (but not if glucose also is present). Catabolite activator protein (CAP) binds cAMP, activates, and binds to a CAP recognition site upstream of the promoter (cAMP is greatly reduced in presence of glucose). CAP changes the conformation of DNA and facilitates binding of RNA polymerase and transcription. When glucose and lactose are present, E. coli preferentially uses glucose due to low levels of active CAP (low cAMP). Adding cAMP to cells restore transcription of the lac operon even when glucose is still present.

20. Fig. 17.11, Positive control of the lac operon with CAP

21. Sequence of the lac operon was the first well-characterized molecular model of gene regulation: lac operon promoter begins at -84 bp immediately after the lacI stop codon and ends at -8 bp from the transcription start site. CAP-cAMP binding site occurs at -54 to -58 and -65 to -69. RNA polymerase binding site spans -47 to -8. Operator is next to the promoter at -3 to +21. mRNA transcript begins at +1 bp within the operator. -galactosidase gene has a leader sequence before the start codon. -galactosidase start codon (AUG) is at position +39 to +41

22. Fig. 17.14, Base pair sequence of controlling sites, promoter, and operator for lac operon of E. coli.

23. The Trp operon of E. coli: If amino acids are present in the growth medium E. coli will “import” amino acids before it makes them. Genes for amino acid synthesis are repressed, repressible operons. When amino acids are absent in the growth medium, genes are “turned on” (or expressed) and amino acid synthesis occurs. The tryptophan (Trp) operon of E. coli is one of the most extensively studied repressible operons.

24. The Trp operon of E. coli; first characterized by Charles Yanofsky et al.: Trp operon spans ~7 kb and produces 5 gene products required for synthesis of the amino acid tryptophan. Trp operon contains 5 biosynthetic coding genes, trpA-E. Promoter and operator are upstream of trpE. Leader region (trpL) occurs between trpA-E coding genes and the operator. Within trpL is an attenuator region (att). TrpR (repressor protein gene) occurs upstream of the promoter.

25. Fig. 17.15, General organization of the Trp operon of E. coli:

26. Regulation of the trp operon: Two mechanisms regulate the trp operon: 1.Repressor/operator interaction 2.Termination of initiated transcripts

27. Regulation of the trp operon: 1. Repressor/operator interaction When tryptophan is present, tryptophan binds to trpR gene product. trpR protein binds to the trp operator and prevents transcription. Repression reduces transcription of the trp operon ~70-fold.

28. Regulation of the trp operon: 2. Termination of initiated transcripts Transcription also is controlled by attenuation, process of translating a short, incomplete polypeptide. When cells are starved for tryptophan, trp genes are expressed maximally. Under less severe tryptophan starvation, trp genes are expressed at lower than maximum levels. Attenuation can regulate transcription level by a factor of 8 to 10, and combined with the repression mechanism, 560-700 fold.

29. Molecular model for attenuation: Recall that a leader region (trpL) occurs between the operator and the trpE sequence. Within this leader is the attenuator sequence (att). att sequence contains a start codon, 2 Trp codons, a stop codon, and four regions of sequence that can form three alternative secondary structures. Secondary structureSignal Paired region 1-2pause Paired region 2-3anti-termination Paired region 3-4termination

30. Fig. 17.16, Organization of the leader/attenuator trp operon sequence.

31. Molecular model for attenuation (cont.): Recall that transcription and translation are tightly coupled in prokaryotes and occur simultaneously. Pairing of mRNA regions 1 and 2 causes RNA polymerase to pause just after these regions are synthesized. Pause is long enough for a ribosome to load onto the mRNA and begin translating just behind RNA polymerase.

32. Molecular model for attenuation (cont.): Position of the ribosome plays an important role in attenuation: When Trp is scarce or in short supply (and required): 1.Trp-tRNAs are unavailable, ribosome stalls at Trp codons and covers attenuator region 1. 2.Region 1 cannot pair with region 2, instead region 2 pairs with region 3 when it is synthesized. 3.Region 3 (now paired with region 2) is unable to pair with region 4 when it is synthesized. 4.RNA polymerase continues transcribing region 4 and beyond synthesizing a complete trp mRNA.

33. Molecular model for attenuation (cont.): Position of the ribosome plays an important role in attenuation: When Trp is abundant (and not required): 1.Ribosome does not stall at the Trp codons and continues translating the leader polypeptide, ending in region2. 2.Region 2 cannot pair with region 3, instead region 3 pairs with region 4. 3.Pairing of region 3 and 4 is the “attenuator” sequence and acts as a termination signal. 4.Transcription terminates before the trp synthesizing genes are reached.

34. Fig. 17.17a, Attenuation model in Trp starved cells.

35. Fig. 17.17b, Attenuation model in Trp non-starved cells.

36. Fig. 17.19, Predicted amino acid sequences of attenuators for Phe, His, Leu, Thr, and Ile operons in E. coli.