# Test 2, Lecture 8 temperate phages can do this choose between lytic and lysogenic multiplication cycles 90% of known phages are... lysogenic Lambda's genome dsDNA; 48 kb; linear in virion, circular in host cells Lambda can circularize inside host cells because of this "cos" sites (cohesive ends). The linear dsDNA has ssDNA overhangs which are complementary, so the ends can base-pair in the presence of DNA ligase. Lambda tail fiber attach to this maltose receptor (lamB) Approximate number of genes in lambda 50 Lambda's genome has this organizational feature Genes are clustered by function in operons Lambda's regulatory genes are in this location Immunity region In Lambda's early gene expression, these promoters are available and they produce these P_L gives N protein, P_R gives cro repressor Lambda's N protein is this and does this An anti-termination factor. It promotes read-through of t_L1 (from P_L, which give CIII, xis, int) and read-through of t_R1 (from P_R, which give CII, O, P) Lambda's O and P proteins are... Homologs of DNA A and DNA C Lambda's Cro protein is this and does this A repressor, which binds an inactivates P_L and P_R. (It is also feedback-inhibited, so it shuts off production of itself as well as CIII, Xis, Int, CII, O, P). In Lambda's delayed early expression, most products are for this lysogeny In Lambda's delayed early expression, these products are for lysogeny and have these functions CII (activator of lysogenic promoter), CIII (stabilize CII), Xis (excisionase required for excision from host chromosome), Int (integrase required for integration into host chromosome). In Lambda's delayed early expression, these products are for the lytic cycle O (homologous to DNA A protein; initiation at 9-mer repeats) P (homologous to DNA C protein; delivers DNA B (helicase) to replication fork). In Lambda's late expression, this occurs Cro represses P_L and P_R, so P_R' is available for producing assembly products Lambda's DNA replication Both theta style and rolling circle; double strands from the rolling circle are packaged into phage heads; nuclease recognizes cos site and cuts packed DNA These Lambda genes code for lysis proteins S, R, and R_S; all are P_R' products In Lambda, this is the key to establishing the lytic phase The activity of the Cro protein. If Cro wins, lysis occurs. # Test 2, Lecture 9 This is responsible for integration of the Lambda DNA into the host chromosome Lambda's integrase complexed with IHF (integration host factor) This is required for integration of Lambda DNA to occur Integrase must be produced (from the P_I promoter which is located in xis), IHF needed to complex with integrase, and P_I must be activated by CII (which in turn is stabilized by CIII) Lambda's CII is required for this Activation of P_I so integrase is produced; activation of P_RE so CI is produced Lambda's CI is this and does this Called the "Lambda Repressor"; turns off all promoters except P_RM (promoter of repressor maintenance). It shuts off all genes except its own gene, so it essentially activates P_RM. In Lambda, this is the key to establishing lysogeny CII In Lambda, this decides whether lytic cycle or lysogeny occurs Relative concentration of Cro (lysis) and CII (lysogeny). If Cro wins, it binds to operators at P_L and P_R, and if CII wins it activates P_RE (and CI and Cro compete directly at O_R). In Lambda, high MOI leads to... lysogeny In Lambda, CII binds as... an oligomer, so high MOI leads to lysogeny In Lambda, for lysogeny to occur, CII is required to be... present in high concentration Lambda's CII is unstable and is degraded by this HflA (High frequency of lysogeny host protein) Since Lambda's CII is degraded by HflA... If the cell's gene for HflA is damaged, then CII builds up in concentration and leads to high frequency of lysogeny This protects Lambda's CII from degradation CIII protects from HflA degrading CII When nutrition of Lambda's host is poor... cAMP increases, which causes HflA to decrease, which causes CII to increase and lead to lysogeny In Lambda, lysogeny is chosen initally if the newly infected host is in poor health because... The lytic cycle is energetically expensive Once in lysogeny, upon these conditions Lambda can do this Poor host health promote induction from lysogeny This host protein serves as a mediator of Lambda lysogeny cAMP. Low glucose increases cAMP concentration; cAMP represses HflA, so CII builds up quickly and promotes lysogeny This represses HflA cAMP Regarding the operator for P_R, this has the highest affinity for this portion of the operator Cro binds OR3 with highest affinity The operator for P_R... OR is divided into 3 binding sites, OR1, OR2, OR3. A general description of an OR region 17 base palindromes with slightly different sequences (which vary binding affinity) Cro binding of the OR region It binds OR3 with highest affinity, OR2 and OR1 with equal but less affinity. CI binding of the OR region It binds OR1 with highest affinity, OR2 with next highest, and OR3 with lowest. CI bound to OR1 and/or OR2 has this effect P_R off (turns of Cro), P_RM stimulated Cro bound to OR3 has this effect P_RM off (turns off CI), P_R on Cro bound to OR1, OR2, and OR3 has this effect All promoters off CI bound to OR1, OR2, and OR3 has this effect All promoters off Cro and CI have these things in common Both recognize palindromes; both bind as dimers; both use helix-turn-helix motif to bind the major groove of DNA Lambda lysogeny is maintained by this CI repressor; keeps all genes repressed except CI gene Lambda superinfection protection is provided by this CI; it represses any invading Lambda DNA To induce Lambda from lysogeny, this must be destroyed CI; so excisionase and integrase are expressed Describe the structure of the CI dimer It has 2 globular domains with a thin linker between them; domain 1 (N-term) is for DNA binding; domain 2 (C-term) is for dimerization The CI dimer is cleaved by this RecA, which is a host SOS protein Induction from Lambda lysogeny RecA, a host SOS response protein, cleaves the CI dimer, leaving one domain bound to DNA. CI can now no longer bind, and since CI was maintaining lysogeny, induction can now commence (excisionase and integrase can now be expressed).