1. L1 The lac operon L2 The trp operon L3 Transcriptional regulation by alternative σ factors alternative σ factors Section L: Regulation of transcription in prokaryotes

2. L1:The LAC Operon Operon - what is it? The operon is a unit of gene expression and regulation The structural genes (any gene other than a regulator) for enzymes involved in a specific biosynthetic pathway whose expression is co-ordinately controlled. Control elements such as an operator sequence, which is a DNA sequence that regulates transcription of the structural genes. Regulator gene(s) whose products recognize the control elements, for example a repressor which binds to and regulates an operator sequence.

3. The structure of operon

4. (Pasteur Institute, Paris, France) Studied the organization and control of the lac operon in E. coli. Earned Nobel Prize in Physiology or Medicine 1965. Francois Jacob and Jacques Monod

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) An enzyme responsible for hydrolysis of lactose to galactose and glucose. Permease (lacY) An enzyme responsible for lactose transport across the bacterial cell wall. Acetylase (lacA) Function is not understood.

6. E. Coli cells need an enzyme to break the lactose down into its two component sugars: galactose and glucose. The enzyme that cuts it in half is called  -galactosidase.  -galactosidase and structure of lactose

7. Lac operon  -galactosidase Permease Acetylase DNA lacI:promoter-lacI-terminator operon:promoter-operator-lacZ-lacY-lacA-terminator Structure of the lac operon

8. Lac operon DNA Regulation genes transcript translate mRNA of repressor Inactive lac repressor No structure genes expression Without inducer-no structure genes expression  -galactosidase PermeaseAcetylase

9. Binding of inducer inactivates the lac repressor

10. cAMP receptor protein The P lac promoter is not a strong promoter. P lac and related promoters do not have strong -35 sequences and some even have weak -10 consensus sequences. For high level transcription, they require the activity of a specific activator protein called cAMP receptor protein (CRP). CRP may also be called catabolite activator protein or CAP. Glucose reduces the level of cAMP in the cell. When glucose is absent, the levels of cAMP in E. coli increase and CRP binds to cAMP.So, the CRP-cAMP complex binds to the lactose operon.

11. CAP-cAMP binding to the lac activator-binding site recruits RNA polymerase to the adjacent lac promoter to form a closed promoter complex. This closed complex then converts to an open promoter complex. CAP-cAMP bends its target DNA by about 90° when it binds. And this is believed to enhance RNA polymerase binding to the promoter, enhancing transcription by 50-fold. DNA-bending and Transcription regulation

12. 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. 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 operons in amino acids synthesis. first characterized by Charles Yanofsky et al. L2 The TRP operon

13. Structure of the trp operon and function of the trp repressor A gene product of the separate trpR operon, the trp repressor, specifically interacts with the operator site of the trp opseron. The symmetrical operator sequence, which forms the trp repressor-binding site, overlaps with the trp promoter sequence between bases -12 and +3. transcription stop site attenuator Trp repressor Active trp repressor Enzyme for tryptophan synthesis tryptophan trpR

14. The attenuator At first, it was thought that the repressor was responsible for all of the transcriptional regulation of the trp operon. However, it was observed that the deletion of a sequence between the operator and the trpE gene coding region resulted in an increase in both the basal and the activated level if transcriptio. This site is termed the attenuator and it lies towards the end of transcribed leader sequence of 162 nt that precedes the trpE initiator codon. The attenuator is a rho-independent terminator site which has a short GC-rich palindrome followed by eight successive U residues. If this sequence is able to form a hairpin structure in the RNA transcript, then it acts as highly efficient transcription terminator and only a 140bp transcript is synthesized.

15. Leader RNA structure and leader peptide hairpins 14aa Pause Anti-termination Termination

16. 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.

17. 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.

18. The presence of tryptophan gives rise to a 10-fold repression of trp operon transcription through the process of attenuation alone. Combined with control by the trp repressor (70-fold), thus means that tryptophan levels exert a 700-fold regulatory effect on expression from the trp operon. Attenuation occurs in at least six operons that encode enzyme concerned with amino acid biosynthesis. Importance of attenuation

19. L3 Transcriptional regulation by alternativeσfactors σ factors appear to be bifunctional proteins that stimultaneously can bind to core RNA polymerase and recognize specific promoter sequence in DNA. Many bacteria, including E. coli, produce a set ofσfactors that recognize different sets of promoters. Some environmental conditions require a massive change in the overall pattern of gene expression in the cell. Under such circumstances, bacteria may use a different set of σ factors to direct RNA polymerase binding to different promoter sequences.

20. Promoter recognition The binding of an alternative σfactors to RNA polymerase can confer a new promoter specificity on the enzyme responsible for the general RNA synthesis of the cell. Comparisons of promoters activated by polymerase complexed to specific σfactors show that each σfactor recognizes a different combination of sequences centered approximately around the -35 and -10 sites. It seems likely that σfactors themselves contacet both of these regions, with the -10 region being most important. The σ 70 subnuit is the most common σfactor in E. coli which is responsible for recognition of general promoters which have consensus -35 and -10 elements.

21. Comparison of the heat-shock (σ 32 ) and general (σ 70 ) responsive promoters Heat shock promoter When E. coli is subjected to an increase in temperature, the synthesis of a set of around 17 proteins, called heat-shock proteins, is induced. The promoters for E. coli heat-shock proteins-encoding genes are recognized by a unique form of RNA polymerase holoenzyme containing a variant σfactor σ 32, which is encoded by the rpoH gene.

22. Sporulation in Bacillus subtilis Vegetatively growing B.subtilis cells from bacterial spores(see Topics A1) in response to a sub-optimal environment. The RNA polymerase in B.subtilis is functionally identical to that in E.coli. The vegetatively growing B.subtilis contains a diverse set of σfactors. Sporulation is regulated by a further set of σfactors in addition to those of the vegetative cells. Different σfactors are specifically active before cell partition occurs, in the forespore and in the mother cell. Cross-regulation of this compartmentalization permits the forespore and mother cell to tightly co-ordinate the differentiation process.

23. Bacteriophage σfactors Some bacteriophages provide new σsubunits to endow the host RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes(e.g. phage T4 in E.coli and SPO1 in B.subtilis). This stragety is an effective alternative to the need forfor the phage to encode its own complete polymerase(e.g. bacteriophage T7,see Topic K2). The B.subtilis bacteriophage SPO1 expresses a ‘cascade’ of σfactors in sequence to allow its own genes to be transcribed at specific stage during virus infection. Initially, early genes are expressed by normal bacterial holoenzyme. Among these early genes is the gene encoding σ 28, which then displaces the bacterial σfactor from the RNA polymerase. The σ 28 -containing holoenzyme is then responsible for expression of the middle genes. The phage middle genes include genes 33 and 34 which specificy a further σ factor that is responsible for the specific trancription of late genes. In this way, the bacteriophage uses the host’s RNA polymerase machinery and expresses its genes in a defined sequential order.