Regulation of Gene Expression in Prokaryotes

1. Regulation of Gene Expression in Prokaryotes Sections 16.1-16.8

2. Gene Regulation • The growth and division of bacteria are regulated by genes • Constitutive Genes-genes that are always active in growing cells • They are essential to the normal functioning of a growing and dividing cells

3. Gene Regulation • Regulated Genes-genes whose activity is controlled in response to the needs of a cell or organism • Several mechanisms have evolved to turn genes on and off depending on the cells metabolic need for the gene products

4. Gene Regulation Inducible (Adaptive) Systems-enzymes are produced when specific chemical substrates are present • Inducer-the regulatory substance that brings about gene induction; help control the expression of regulated genes • Transcription of an inducible gene occur when a regulatory protein is present. • When a regulatory protein is present the gene is turned on, mRNA is produced, and the gene is produced • Ex: inducible lac genes-gene activity is induced when lactose is added to the medium

5. lac Operon • Operon- a cluster of genes, the expressions of which are regulated together by operator-repressor protein interactions plus the operator region itself and the promoter • Three structural genes in the lac operon • lacZ (Beta- galactosidase)-breaks down lactose into glucose and galactose; it catalyzes the isomerization of lactose to allolactose • Allolactose not lactose is the inducer molecule responsible for the increased production of the three enzymes • lacY (Lactose permease)-actively transports lactose into the cell • lacA (Transacetylasae)-the function of this enzyme is poorly understood, but it may be involved in the removal of toxic by-products of lactose digestion from the cell. • All three genes are transcribed as a single polycistronic mRNA • The order of the controlling elements and genes in the lac operon is lacI-promoter-operator-lacZ-lacY-lacA

6. lac Operon

7. lac Operon • Negative control of the lac operon occur when the repressor is bound to the operator, RNA polymerase can bind to the operon’s promoter but is blocked from initiating transcription • Even in the absence of the inducer a low level of transcription still occur because when the repressor unbinds and before another binds, an RNA polymerase could initiate transcription of the operon

8. lac Operon

9. Jacob and Monod • Jacob and Monod isolated mutants in which all the enzymes of the operon were synthesized constitutively (regardless of the presence or absence of the inducer) • Two types of constitutive mutations: • Operator (lacO) • Repressor gene (lacI)

10. Operon Mutations • Operator constitutive mutations (lacOc)-base pair alterations of the operator DNA sequence make it unrecognizable to the repressor protein • The repressor cannot bind and the genes are constitutively expressed • Cis-dominant • The lacI gene mutations regulate transcription on the structural genes by producing a repressor molecule • The repressor reversibly interacts with another molecule causing a conformational change (allosteric shift) • As a result, the repressor loses its affinity for the lac operator and dissociates from the site • With no repressor bound to the operator, RNA polymerase initiates synthesis of the genes • Trans-dominant • Superrepressor (Is) • No production of lac enzymes in the presence or absence of lactose; transcription of the genes never occur • The superrepressor protein can bind to the operator, but cannot recognize the inducer allolactose • Trans-dominant

11. Constitutive Mutations

12. Superrepressor

13. Positive Control • The positive control system turn on the expression of the operon ensuring that the lac operon will be expressed at high levels ONLY if lactose is the sole carbon source and NOT if glucose is present as well. • If only lactose is present the positive regulation of the lac operon will occur • Catabolite activator protein (CAP) binds with cAMP to form a CAP-cAMP complex • CAP-cAMP complex binds to the CAP site, which is upstream of the site at which RNA polymerase binds to the promoter. • This binding facilitates binding of the RNA polymerase and the initiation of transcription

14. Positive Control • When glucose is in the medium along with lactose, the glucose is used because catabolite repression occurs • The lac operon is expressed at low levels even though lactose is present in the medium • Glucose causes the amount of cAMP in the cell to be greatly reduced • As a result, insufficient CAP-cAMP complex is available to facilitate RNA polymerase binding the lac promoter even though repressors are removed from the operator by the presence of allolactose

15. Positive Regulation

16. Gene Regulation Repressible Systems-the presence of a specific molecule inhibits genetic expression • Gene activity is repressed when a chemical is added • Ex: trp operon-gene activity is repressed when tryptophan is present • If a sufficient amount of tryptophan is present it is energetically inefficient for the organism to synthesize the enzymes necessary for tryptophan production

17. trp operon • Five structural genes A-E • The promoter and the operator regions are upstream from trpE • Between the promoter-operator and trpE is a short leader region, trpL • With in trpL is an attenuator site that plays an important role in the regulation of the trp operon

18. Regulation of the trp operon • The regulatory gene for the trp operon is trpR • The product of trpR is an corepressor protein, which is an inactive repressor that alone cannot bind to the operator • When tryptophan is abundant within the cell, it interacts with the corepressor and converts it to an active repressor • The active repressor binds to the operator and prevents the initiation of transcription of the trp operon • In the presence of excess tryptophan a hairpin loop is formed that behaves as a terminator structure • As a result the tryptophan enzymes are not produced

19. Terminator Hairpin

20. Regulation of the trp operon • The second regulatory mechanism occur when tryptophan is limited • Under severe tryptophan starvation the trp genes are expressed • If tryptophan is scarce an antiterminator hairpin loop is formed • As a result transcription is allowed to proceed past the involved DNA sequence and the entire mRNA is produced

21. Antiterminator Hairpin

22. trp Operon