REVIEW SESSION

1. REVIEW SESSION Wednesday, September 15 5:30 PM SHANTZ 242 E

2. Gene Regulation

3. Gene Regulation Gene expression can be turned on, turned off, turned up or turned down! For example, as test time approaches, some of you may note that stomach acid production increases dramatically…. due to regulation of the genes that control synthesis of HCl by cells within the gastric pits of the stomach lining.

4. Gene Regulation in Prokaryotes Prokaryotes may turn genes on and off depending on metabolic demands and requirements for respective gene products. NOTE: For prokaryotes, “turning on/off” refers almost exclusively to stimulating or repressing transcription

5. Gene Regulation in Prokaryotes Inducible/Repressible gene products: those produced only when specific chemical substrates are present/absent. Constitutive gene products: those produced continuously, regardless of chemical substrates present.

6. Gene Regulation in Prokaryotes Regulation may be Negative: gene expression occurs unless it is shut off by a regulator molecule or Positive: gene expression only occurs when a regulator mole turns it on

7. Operons In prokaryotes, genes that code for enzymes all related to a single metabolic process tend to be organized into clusters within the genome, called operons. An operon is usually controlled by a single regulatory unit.

8. Regulatory Elements cis-acting element: The regulatory region of the DNA that binds the molecules that influences expression of the genes in the operon. It is almost always upstream (5’) to the genes in the operon. Trans-acting element: The molecule(s) that interact with the cis-element and influence expression of the genes in the operon.

9. The lac operon The lac operon contains the genes that must be expressed if the bacteria is to use the disaccharide lactose as the primary energy source. To be used as an energy source, lactose must be cleaved into glucose and galactose. The glucose is then available for metabolism (glycolysis). Note: glucose is the preferred energy substrate.

10. Negative Control The genes in the lac operon are normally turned off, and only expressed when a repressor molecule is removed from the regulatory region. This repressor is removed only in the presence of lactose

11. LacI P O lacZ lacY lacA The lac Operon Regulatory Region Repressor gene Structural Genes P=Promoter O=Operator

12. Structural Genes Structural genes are those that encode for the enzymes that do the metabolic work. LacZ: b-galactosidase, cleaves lactose into glucose and galactose LacY: Permease, promotes entry of lactose into cell LacA: Transacetylase, thought to reduce toxicity of byproducts of lactose metabolism

13. Structural Genes In prokaryotes, all the structural genes within an operon are usually transcribed as a single mRNA, then the genes are independently translated by ribosomes.

14. LacI P O lacZ lacY lacA LacI—The Repressor LacI is the regulatory molecule. When there is no lactose present in the cell,LacI binds to the Operator element and blocks binding of RNA polymerase to the Promoter element. X

15. LacI—The Repressor When lactose IS present, the genes to metabolize lactose must be expressed. Lactose itself causes LacI to dissociate from the operator, which frees up the promoter region, allowing RNA polymerase to bind, and transcription begins. Lactose is the inducer molecule for the lac operon.

16. LacI P O lacZ lacY lacA Induction of the lac operon Lactose Binding of lactose causes a change in the shape of LacI

17. LacI LacI P P O O lacZ lacZ lacY lacY lacA lacA Induction of the lac operon

18. What happens if you mutate LacI? LacI encodes the lac repressor, which keeps the operon shut off in the absence of lactose.

19. What happens if you mutate LacI? Inactivation of LacI would be called a constitutive mutation, because the genes of the lac operon would be on all the time even if there is no lactose present (removed repression).

20. LacI P O lacZ lacY lacA Positive Control of the lac Operon A further increase in transcription of the lac operon occurs if a molecule called catabolite-activating protein(CAP) also binds the promoter region. CAP facilitates the binding of RNA polymerase, and therefore increases transcription

21. LacI P O lacZ lacY lacA Positive Control Remember, glucose is the preferred substrate. CAP exists in the state that will bind the promoter ONLY when glucose is absent. This is the form CAP takes when there is no glucose

22. LacI P O lacZ lacY lacA Positive Control When glucose is present, CAP exists in a state that will NOT bind the promoter of the lac operon. X This is the shape CAP takes when glucose is present. It cannot bind the promoter in this shape X

23. Regulation of the lac Operon So, transcription is regulated as follows: Off when lactose is absent (repressed) Active when lactose is present as well as glucose (de-repressed) Really active when lactose is present but glucose is absent (activated)

24. Gene Regulation in Eukaryotes

25. Differences between Prokaryotes and Eukaryotes 1. DNA is a lot more complicated in eukaryotes—there’s a lot more of it and it’s complexed with proteins to form chromatin 2. Genetic information is carried on multiple chromosomes 3. Transcription and translation are physically separated

26. Differences between Prokaryotes and Eukaryotes (cont.) 4. Eukaryotic mRNA is processed prior to translation 5. Eukaryotic mRNA is much more stable (not as easily degraded) Gene expression can be controlled at the level of translation! 6. Different cell types express different genes

27. Chromatin Remodeling Chemical alteration of the histone proteins of chromatin facilitates or inhibits access of RNA polymerases to DNA promoters.

28. Recruitment of Co-activators Remember enhancer elements? These are binding sites for molecules that influence formation of the RNA polymerase initiation complex. Enhancer elements may have DNA sequences for both positive and negative regulators of transcription.

29. Enhancers The presence or absence of regulators is determined by the cell’s environment, metabolic state, developmental state and/or the presence or absence of signal molecules. The net effect of all the information available, summed up by the regulators present, dictates the transcription efficiency of RNA polymerase from a given promoter.

30. DNA Methylation Chemical modification of DNA by adding or removing methyl (-CH3) groups from the DNA bases, usually cytosine. The presence of the methyl group alters the shape of DNA, which influences the binding of proteins to the methylated DNA.

31. DNA Methylation Typically, increased methylation decreases transcription efficiency. In mammalian females, one X chromosome is inactivated (only one of the X chromosomes is used to drive transcription). The inactivated X chromosome has much more methylation than the active chromosome.

32. Post-Transcriptional Regulation Alternative Splicing: Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 1. Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 2. Exon 1 Exon 2 Exon 4 Exon 5

33. Post-Transcriptional Control RNA Stability • Stability sequences • Instability sequences • Translation efficiency—increased translation increases stability