1. 9-1 Chapter 9 DNA-Protein Interactions in Bacteria Student learning outcomes: Describe examples of structure /function relationships in phage repressors Appreciate that altered specificity repressors and operator mutants clarify mechanisms of amino acid: base pair recognition Impt. Figs. 1*, 2, 3, 4, 6, 7, 8, 14, 16, 17 Q: 1, 2, 3, 4, 5, 7, 10, 11 Cro binding DNA

2. 9-2 The Family of Repressors:  434, P22 Repressors have recognition helices that lie in major groove of appropriate operator Helix-turn-helix motif (HTH) Specificity of bp binding depends on amino acids in recognition helices Phages are not immune to super-infection by each other Fig. 2

3. 9-3 Binding Specificity of lambda-like Repressor: Operator DNA Fig. 1 Recognition helices fit sideways in major groove of operator DNA Certain amino acids on DNA side of recognition helix 2 make specific contact with bases in operator Contacts determine specificity of protein-DNA binding ** Changing amino acids can change specificity of repressor to different DNA sequence

4. 9-4 Probing Binding Specificity by Site- Directed Mutagenesis - Mark Ptashne Fig. 3 Fig. 4 DNase footprint Key amino acids in recognition helices of P22, 434 repressors proposed Amino acids differ between repressorss Change 5 aa of 434 to P22; see altered specificity repressor binds P22 DNA

5. 9-5 Repressor  repressor has extra motif, N- terminal arm that aids binding by embracing DNA Cro and repressors share affinity for same operators, but micro- specificities for O R 1(l) or O R 3 (cro) Specificities determined by interactions between different amino acids in recognition helices and different base pairs in operators Fig. 4 repressor dimer on OR2

6. 9-6 High-Resolution Analysis of Repressor-Operator co-crystal –Recognition helices (3 red) of each monomer nestle into DNA major grooves (2 half-sites) –Helices hold two monomers together in repressor dimer –DNA is similar to B-form DNA –DNA bends at ends of fragment as curves around repressor dimer Fig. 6; operator sequence; Fig. 7 model

7. 9-7 Amino acids of repressor hydrogen bond with Bases in major groove Fig. 8

8. 9-8 Amino Acid: DNA Backbone Interactions Hydrogen bond at Gln33 maximizes electrostatic attraction between positively charged amino end of  -helix and negatively charged DNA Attraction works to stabilize bond Fig. 9

9. 9-9 High-Resolution Analysis of 434 Repressor-Operator Interactions Genetic and biochemical data predicted R-O contacts X-ray crystallography of 434 repressor-fragment/ operator- fragment shows H bonding at Gln residues in recognition helix to 3 bp in DNA Potential van der Waals contact between Gln29 and 5Me of T3 Fig. 10

10. 9-10 Phage 434: Effects on DNA Conformation R-O complex DNA deviates from normal shape DNA bends to accommodate base /aa contacts Central part of helix is wound extra tightly Outer parts are wound more loosely than normal DNA sequence of operator facilitates bending Fig. 11 Normal DNA; DNA bent by 434 repressor binding

11. 9-11 9.2 trp Repressor and role of Tryptophan trp repressor uses helix-turn-helix (HTH) DNA binding motif to contact operator Aporepressor is not active in binding DNA Tryptophan forces recognition helices of trp repressor dimer into proper position to bind trp operator Fig. 12

12. 9-12 9.3 General Considerations on Protein-DNA Interactions; multimeric proteins Specificity of binding between protein and specific stretch of DNA relates to: –Specific interactions between bases and amino acids –Ability of DNA to assume shape that directly relates to DNA’s base sequence Target sites for DNA-binding proteins usually symmetric or repeated Most DNA-binding proteins are dimers: greatly enhances binding between DNA and protein as protein subunits bind cooperatively

13. 9-13 Hydrogen Bonding Capabilities of Different Base Pairs Protein ‘reads the DNA’ Different base pairs present four different hydrogen-bonding profiles to amino acids approaching either major or minor groove Fig. 14

14. 9-14 9.4 DNA-Binding Proteins: Action at a Distance DNA-binding proteins can influence interactions at remote sites in DNA – often looping intervening DNA Common in eukaryotes Occurs in several prokaryote systems : lac operon multiple operators ara operon looping gal operon looping repressor

15. 9-15 E. coli gal Operon gal operon has 2 operators, 97 bp apart –One adjacent to gal promoter - External operator, O E –Other located within first structural gene, galE O i 2 separated operators - both bind repressors that interact, loop out intervening DNA Recall Chapt. 7 lacI, araC repressors Fig. 15

16. 9-16 DNA Looping affects DNase Susceptibility Operators separated by –Integral number of double- helical turns loop out DNA to allow cooperative binding –Nonintegral number of turns requires proteins to bind opposite faces of DNA, no cooperative binding Fig. 16 Fig. 17 repressor binds cooperatively to operators

17. 9-17 Enhancers Enhancers are nonpromoter DNA elements that bind protein factors and stimulate transcription –Can act at a distance –Originally found in eukaryotes (lots chapt. 12) –Recently found in prokaryotes. E. coli glnA gene: NtrC protein binds enhancer, Binds RNAP 70 bp away NtrC hydrolyzes ATP, lets RPo form. Insert 350 bp, see loop Fig. 20 NtrC: RNAP

18. Review questions 1. Draw rough diagram of helix-turn-helix domain interacting with DNA double helix 2. Describe experiment that shows which amino acids bind which base pairs in -like phage repressors. 10. Explain fact that protein oligomers (dimers, tetramers) bind better to DNA than monomeric proteins 9-18