Gene regulation II Biochemistry 302. Bob Kelm March 1, 2004

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1 Gene regulation II Biochemistry 302 Bob Kelm March 1, 2004

2 Lessons to learn from bacteriophage λ in terms of transcriptional regulation Similarities to E. coli Cis-elements (operator elements) are adjacent to the genes they control. Binding by regulatory proteins (trans-acting factors) leads to activation or repression. Differences from E. coli (virus-host relationship) Control mechanisms are more subtle and complex. Mechanisms must accommodate viral life cycle.

3 Phage λ: Choice of lysis or lysogeny based on regulated gene expression E. coli-phage λ hybrid chromosome E. coli chromosome Step 1: infection

4 Possible outcomes of λ viral infection: lysis (replicate and kill host) or lysogeny Lysogeny established by circularization of phage λ DNA then site-specific recombination into E. coli host chromosome. Dormant phage maintained as transcriptionally repressed prophage. De-repression leads to excision of circular phage DNA, replication of phage genome, and activation of genes required for viral particle assembly. Fig (lysogenic pathway)

5 Distinct patterns of gene expression needed to accommodate λ physiology Infection leading to lytic growth Infection leading to lysogeny Long-term maintenance of lysogeny Breaking of lysogeny lytic growth

Phenotypes of lac and λ mutants help identify operator-repressor system Table 26-2 All the cells are lysed (normally turbid because lysogenized bacteria are immune and

6 Phenotypes of lac and λ mutants help identify operator-repressor system Table 26-2 All the cells are lysed (normally turbid because lysogenized bacteria are immune and continue to grow). Lysogenic mutant mutation in repressor protein (e.g. ci) or in genes controlling its synthesis (e.g. cii/ciii) Virulent mutant mutation in operator DNA

7 Phage λ : Basic features of gene regulatory apparatus Life cycle is based on a choice between lytic versus lysogenic growth. Genome is organized around early and late gene products. Gene expression in phage λ occurs in a coordinated manner following infection Immediate early genes Early genes Late genes Essence of the developmental switch: competing actions of the DNA-binding proteins ci repressor (also called λ repressor) and Cro.

8 Complexity of the phage λ genome: Early genes, late genes, and corresponding regulatory elements RE: establish lysogeny (repressor establishment) RM: maintain lysogeny (repressor maintenance)

9 A closer view of the early gene regulatory region of phage λ Fig Regulatory proteins ci and Cro (repressors but ci can act as an activator as well) cii and ciii (activator/coactivator of gene encoding ci) N (antiterminator interacts with NusA to block termination) Int and Xis (integration and excision), O and P (initiation of replication) Cis-elements Multiple operators Directional promoters (leftward and rightward)

10 An even closer look at the complexity of the O R P R regulatory region of phage λ Fig Three repressor protein-binding sites/operator Leftward (P RM promoter of repressor maintenance, i.e. ci) and rightward (P R controls Cro expression) promoters ( 10 and 35 sites) interspersed with repressor protein-binding sites

11 Protein-binding properties of the O R P R regulatory region of phage λ ci or λ repressor (M. Ptashne, 1967) Dimer, 27 kda subunits (10 7 M in lysogenic state) O R 1 > O R 2 > O R 3 in terms of affinity (Ka M 1 ) Highly cooperative DNA-binding (activates its own transcription under certain conditions) Cro (ci repressor off) Homodimer (66 residues/subunit) O R 3 > O R 2 = O R 1 (but much less tightly than ci) Noncooperative DNA-binding

12 The fate of the viral genome is dictated by competition between lytic (Cro) and lysogenic (cl) gene regulators for occupancy of O R P R P RM region.? When ratio of Cro/cI is large, lysis and/or excision is the favored pathway. X ci transcription cro transcription Fig

13 Real and imagined structures of the Cro dimer-operator complex DNA-binding motif? DNA-binding region? Mislabeled in textbook Fig Fig Distance between #3 helices, significance?

14 Structural basis of ci and Cro operator DNA-binding ci Cro Recognition helices align parallel to base pairs, helix 1 arm contacts consensus operator half-site Recognition helices align parallel to major groove R.A. Albright and B. W. Matthews PNAS 95: , 1998

15 Why ci and Cro vary in their relative affinity and specificity for different operators HTH motif is conserved between ci and Cro repressors but. Amino acid sequences comprising DNA-binding helices are different. Conserved AAs contact invariant nucleotides. Unique AAs contact variant nucleotides. Additional helix 1 arms on ci extent around the DNA duplex making additional base contacts. ci Cro Fig

Schematic of nucleotide contacts by ci and Cro repressors Nonconsensus half-sites vdw O R 1 O R 3 HB 3 5 8 3 5 8 Note: Only hydrogen bonds (HB)

16 Schematic of nucleotide contacts by ci and Cro repressors Nonconsensus half-sites vdw O R 1 O R 3 HB Note: Only hydrogen bonds (HB) and van der Waals (vdw) contacts are highlighted but non- H bond electrostatic interactions w/ bases & phosphate backbone also contribute. Fig

17 Summary of the regulatory mechanism governing the phage λ life cycle λ has two developmental outcomes: lysis or lysogeny. The different outcomes are associated with the expression of different genes. The choice is the result of a competition between two DNA-binding proteins for three different sites. Functional balance between Cro and ci repressors is connected to the physiological state of the cell. In more general terms, eukaryotic cells exploit similar mechanisms to control developmental choices/switches. Proliferation vs differentiation Life vs death (apoptosis)

Chapter 8 DNA Recognition in Prokaryotes by Helix-Turn-Helix Motifs

Chapter 8 DNA Recognition in Prokaryotes by Helix-Turn-Helix Motifs 1. Helix-turn-helix proteins 2. Zinc finger proteins 3. Leucine zipper proteins 4. Beta-scaffold factors 5. Others λ-repressor AND CRO