Seminar W 4 pm BI 234 Dr. K. Cude!!

1. Seminar W 4 pm BI 234 Dr. K. Cude!! A novel NFb pathway in the regulation of theG2/M phase of the cell cycle Refreshments at 3:45 pm….Be there!!!

2. Suggested Readings: Transcription Voet and Voet: Ch 31 Problems: 8, 11, 12, 13, 15 Translation: Voet and Voet Ch 32 Problems: 8, 12, 13, 14, 17, 19, 20, 21 Watson Ch 14

4. Regulation Where can it happen? How? Why??

11. Figure 31-1 The induction kinetics of b-galactosidase in E. coli. Page 1217

12. The lac operon • E-coli uses three enzymes to take up and metabolize lactose. • The genes that code for these three enzymes are clustered on a single operon – the lac Operon. What’s lactose??

13. Figure 31-2 Genetic map of the E. coli lac operon. Page 1218

15. Element purpose Operator (LacO) binding site for repressor Promoter (LacP) binding site for RNA polymerase Repressor (LacI) gene encoding lac repressor protein Binds to DNA at operator and blocks binding of RNA polymerase at promoter Pi promoter for LacI CAP binding site for cAMP/CAP complex The Lac Operon

16. Figure 31-26 The nucleotide sequence of the E. coli lac promoter–operator region.

17. Figure 31-25 The base sequence of the lac operator.

19. The lac repressor gene • Prior to these three genes is an operator region that is responsible for turning these genes on and off. • When there is not lactose, the gene for the lac repressor switches off the operon by binding to the operator region. • A bacterium’s prime source of food is glucose. • So if glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism.

22. Figure 31-36 X-Ray structure of the lac repressor subunit. Page 1248

23. Figure 31-37aX-ray structure of the lac repressor-DNA complex. Page 1249

26. Induction. • Allolactose is an isomer formed from lactose that derepresses the operon by inactivating the repressor, • Thus turning on the enzymes for lactose metabolism.

27. The lac operon in action. • When lactose is present, it acts as an inducer of the operon (turns it on). • It enters the cell and binds to the Lac repressor, causing a shape change that so the repressor falls off. • Now the RNA polymerase is free to move along the DNA and RNA can be made from the three genes. • Lactose can now be metabolized (broken down).

28. When the inducer (lactose) is removed • The repressor returns to its original shape and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made. • Note that RNA polymerase can still bind to the promoter though it is unable to move past it. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription.

29. Lac movie Lac and trp

30. Lac repressor induces major conformational changes in DNA http://molvis.sdsc.edu/atlas/morphs/lacrep/lacrep_anim_large.gif

32. Catabolite repression happens when glucose (a catabolite) levels are high. • Then cyclic AMP is inhibited from forming. • When glucose levels drop, more cAMP forms. • cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. • This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. • This phenomenon is called catabolite repression,

33. Suggested readings on regulation/dna bp Voet pp 1237-1253 Problems 2, 4 Here’s a quiz on the lac operon: http://www.bio.davidson.edu/courses/movies.html

34. MVA Fig. 26.35

36. MVA Fig. 26.36

38. Figure 31-41 The alternative secondary structures of trpL mRNA. Page 1252

39. Figure 31-42a Attenuation in the trp operon. (a) When tryptophanyl–tRNATrp is abundant, the ribosome translates trpL mRNA.

40. Figure 31-42b Attenuation in the trp operon. (b) When tryptophanyl–tRNATrp is scarce, the ribosome stalls on the tandem Trp codons of segment 1.

41. Trp operon And again all the cool animation files

42. How does a repressor find its operator in a sea of other sequences? It is not enough just for the regulatory protein to recognize the correct DNA site. The protein must also find it rapidly and bind to it sufficiently tightly to discriminate it from the millions of competing and overlapping nonspecific sites that are explored in the course of specific target localization.

43. One point to keep in mind while considering protein-DNA interactions is that such an interaction represents a dynamic equilibrium: Whether an operator has its (or a) particular repressor protein bound to it depends on: 1. the concentration of the regulatory protein in the cell, 2. the affinity between the repressor and the operator sequence and 3. the affinity between the repressor and other non-specific DNA binding sites.

44. Association constants: lac repressor + DNA to R-DNA complex Repressor: lac operator 1-2 X 1013 M-1 other DNA 2-3 X 106 M-1 (specificity = KA(s)/KA(non-specific) = 107) Repressor bound to inducer lac operator 2 X 1010 M-1--or some references suggest this is even lower other DNA 2 X 106 M-1 When repressor is bound to allosteric regulator (allolactose in this case) non-specific binding competes more effectively with specific binding.

45. How a repressor recognizes and binds to an operator The interaction between repressor and operator is often taken as a paradigm for sequence-specific DNA-protein interactions. Each regulatory protein in E. coli must select its operator site (or sites) from among the five million or so base pairs of DNA in the cell. For this organism, an operator (or any other cis acting site) must be at least 11-12 bases long in order to form a site that reoccurs at random less than once per genome. Accordingly, regulatory proteins in E. coli bind tightly to specific DNA sequences that are about 15-20 base pairs long.

46. How a repressor recognizes and binds to an operator The interaction between repressor and operator is often taken as a paradigm for sequence-specific DNA-protein interactions. Each regulatory protein in E. coli must select its operator site (or sites) from among the five million or so base pairs of DNA in the cell. For this organism, an operator (or any other cis acting site) must be at least 11-12 bases long in order to form a site that reoccurs at random less than once per genome. Accordingly, regulatory proteins in E. coli bind tightly to specific DNA sequences that are about 15-20 base pairs long.

47. Operator Sequence and Structure: A large number of operator sites have been identified and their DNA sequence has been determined. One feature that is common to all operators is an imperfect two-fold axis of symmetry. • A perfectly symmetrical sequence is shown below. • >---- G C C A T G C G C A T G G C ----> • <---- C G G T A C G C G T A C C G ----<

48. Cap binding site: Link to view structure

49. Lac repressor lac operator: binding site for the lac repressor protein (lac I gene product)

50. Structure of Regulatory Proteins: Many DNA-binding regulatory proteins share features in common that reflect a common mode of DNA binding. Some of these features are: (1) The active binding unit is a dimer of two identical globular polypeptide chains oriented oppositely in space to give a molecule with a two-fold axis of symmetry phage lambda cI repressor protein alpha helical region in contact with the major groove is in red.