Bacterial and Phage Genetic Switches

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1 Bacterial and Phage Genetic Switches Prof. C. J. Dorman Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin.

2 Lecture 1 The genetic switch controlling the lytic-lysogen decision in bacteriophage lambda

3 Switch types active Binary (On/Off) inactive Rheostat (set over a spectrum of values) active over a range of activity levels

4 Transcription control: regulating the activity of RNA polymerase α-ntd ββ' α CTD σ UP element Activators Repressors

5 Coordinated control of transcription (I) individual gene operon Stimulus regulon Common regulatory protein

6 Coordinated control of transcription (II) Stimulus 1 Regulatory protein 1 Stimulus 2 Regulatory protein 2

7 Effects of stimulus on the regulator P Covalent modification e.g. phosphorylation Fe Bind ligand (metal, sugar, etc) oligomerize Proteolytic degradation

8 Regulator-regulator interactions Activator displaces repressor inactive active Activator reinforces a second activator active highly active

9 Bacteriophage lambda, a temperate phage Head infection DNA LamB protein Tail

10 Lambda cos sites: genome circularization cos cos cos cos CCCGCCGCTGGA GGGCGGCGACCT GGGCGGCGACCT CCCGCCGCTGGA

11 Summary of the lambda lifecycle: lytic 1. adsorption 2. nucleic acid injection 3. genome circularization 6. lysis 5. maturation 4. latent period

12 Summary of the lambda lifecycle: lysogenic 1. adsorption 2. nucleic acid injection 3. genome circularization 4. recombination LYSOGEN INDUCTION LYSIS

13 Lambda integration: site-specific recombination lambda attp gal attb bio bacterial chromosome Integrase (int gene product) + Integration Host Factor lambda prophage gal attl attr bio

14 Lambda excision: site-specific recombination lambda prophage attl attr gal bio integrase (int) excisionase (xis) + Integration Host Factor lambda attp gal attb bio

15 Lambda genome (48,502 bp) recombination DNA replication Head Tail immunity lysis Nu1 A W B C Nu3 D E FI FII Z U V G T H M L K I lom Ea47 Ea31 Ea59 att int xis Ea8.5 Ea22 exo bet gam kil ciii ssb ral git N rexba ci cro cii O P ren Q S R Rz cos PL cos PM PE PR

16 Transcription post infection and circularization - lytic cycle tl1 PL ciii N ci cro Q S PR tr1 tr2 tr3 PR' FIRST gene products to appear are N and Cro. EARLY GENES N is an anti-terminator. It allows transcription to ignore terminators tl1, tr1, and tr2. ANTI-TERMINATION allows expression of the DELAYED EARLY GENES THEN Q enables transcription from PR'. this transcript encodes proteins for phage head and tail assembly (the LATE GENES). MEANWHILE Cro builds up and represses the PL and PR promoters when they are no longer needed.

17 Establishment of lysogeny PE ciii N PL ci PM cro cii PR The ci repressor switches off the lytic cycle The ci gene is expressed from the promoter for repression establishment, P E. Activity of P E requires the cii activator, an unstable protein. The ciii protein stabilizes cii. Neither cii nor ciii can be expressed until the N anti-terminator is active. Production of cii and ciii allows expression of ci repressor.

18 Effects of ci repressor activity REPRESSION ACTIVATION PE (LOSS OF ACTIVATION) ciii N PL ci PM cro cii PR REPRESSION ci repressor binds to operator sites at the P L and P R promoters and shuts them off. This stops production of N and Cro regulatory proteins, blocking the lytic cycle. HOWEVER, it also shuts off cii and ciii production, needed for ci transcription. Repressor binding has a stimulatory effect on P M, the promoter for repressor maintenance. P M activity keeps ci repressor levels topped up.

19 Cro and ci are competitors REPRESSION by Cro ACTIVATION by ci operator OR ciii N ci PM cro cii PR REPRESSION by Cro REPRESSION by ci Cro and ci compete for binding to the operator sequence between promoters P M and P R. Each is a repressor of P R. But Cro represses P M too. Cro prevents ci synthesis in two ways: 1) by switching off the cii gene and 2) by inhibiting the promoter for ci maintenance.

20 Cro and ci interactions with the operator OR ACTIVE PM ci ci OR3 OR2 OR1 REPRESSED PR REPRESSED PM Cro Cro Cro REPRESSED PR

21 The lytic-lysogenic decision?????? Lysis Lysogeny Binding of ci to OR1 prevents the lytic cycle Binding of Cro to OR3 prevents the maintenance of lysogeny by repressing P M. (Remember, P E will only continue to function for as long as the cii protein survives.)

22 READING MATERIAL "Phage Strategies" (Chapter 27 in Lewin's Genes X by B Lewin [2011] Edited by JE Krebs, ES Goldstein, ST Kilpatrick) Article is available in the Microbiology Departmental Office First floor Moyne Institute of Preventive Medicine