2. Suggested readings on regulation/dna bp Watson pp.480-500, 504-509 Voet pp 1237-1253 Problems 2, 4 Here’s a quiz on the lac operon:quiz http://www.bio.davidson.edu/courses/movies.html Do interactive exercise 34.55

3. Suggested Readings: Transcription Voet and Voet: Ch 31 Problems: 8, 11, 12, 13, 15 Watson: Ch12 pp. 347-362 371-374 Ch 13 Translation: Voet and Voet Ch 32 Problems: 8, 12, 13, 14, 17, 19, 20, 21 Watson Ch 14

4. MVA Fig. 26.35

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

8. Trp operon And again all the cool animation files

9. 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.

10. 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.

11. 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.

12. 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 ----<

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

14. 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.

15. (2) The critical contacts between the protein and the DNA are made by adjacent  helices located at the binding face of each monomer. The helices are connected by a turn in the protein secondary structure. This helix-turn-helix motif is common to many regulatory proteins. HTH

16. BindingBinding motifs http://www.umass.edu/molvis/freichsman/Site642/page_dnab/menu.html again http://www.web-books.com/MoBio/Free/Ch4F2.htm#4F1

17. Cro Why do the recognition helices contact the major groove? What determines the specificity of interaction?

19. A=H bond acceptor D=H bond donor

20. One of the most common DNA-protein interactions. Because of its specific geometry of H-bond acceptors, guanine can be unambiguously recognized by the side chain of arginine

21. Stereo view for phage lambda repressor

23. Specificity of protein-DNA interaction of due to:  ability of amino acid side chains in the recognition helix to form hydrogen bonds with specific bases in its cis-acting site  multiple complementary interactions between the protein and the DNA that are dependent on the deformation of the helix and which increase the number of contact points

24. Main features of interactions between DNA and the helix-turn-helix motif of DNA binding proteins

25. Voet and Voet Interactive exercise 34.55

28. Phage Lambda

29. Binding tutuorialtutuorial http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ Nucleic_Acids/prodna.html

30. Important Points: a handshake leads to a bear-hug Specific recognition of DNA targets by the helix-turn-helix motif involves interactions between sides of the recognition helix and bases in the major groove of the DNA But, specific recognition of DNA sequences is to a large extent governed by other interactions within complementary surfaces between the protein and the \ These interactions frequently involve H-bonds from protein main- chain atoms to the DNA backbone in both the major and the minor groove and are dependent on the sequence-specific deformability of the target DNA

31. DNA deformation induced by protein-binding. The ease with which a stretch of DNA can be deformed can affect the affinity of protein binding to a specific sequence

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

33. CAP binding to its cis-acting site  cAMP binding domain in blue  red -- DNA phosphates whose ethylation interferes with cap binding  blue hypersenstivie to DNase I-- these phosphates bridge the cap-induced where the minor groove has been widened

34. No direct H-bonding with bases! All specific H-bonds occur via bridging water molecules! Only direct contacts are H-bonds to the phosphate backbone!!!! Yet mutations of these non-contact bases alter binding specificity This suggests that the operator assumes a sequence-specific conformation that makes favorable contacts with the repressor known as Indirect Readout

35. When tryptophan is added to crystals of aporepressor, the crystals shatter. When the tryptophan wedges itself into the protein, it changes the shape of the protein enough to break the lattices of the crystal The orientation of the recognition helix shifts when tryptophan is bound.

36. His Cys