Biochemistry 110 Carl Parker x6368 102 Braun Lab Central Dogma of Molecular Biology DNA-‐Dependent RNA Polymerase • • • • • Requires a DNA Template Synthesizes RNA in a 5’ to 3’ direcLon Requires ribonucleoside tri-‐phosphates rNTPs Requires a divalent caLon Mg The enzyme is very large with 4 subunits (bacterial enzyme) Structure of E. coli RNAP Es and Ecore have diﬀerent properLes • Both will transcribe a heterogenous sheared DNA template • Only the holoenzyme can transcribe an intact speciﬁc template • Ecore has the catalyLc acLvity • The sigma subunit plays a role in template selecLon Activity of core vs holoenzyme. Hinkle and Chamberlain dissociation experiment. Closed and Open Promoter Complexes Sigma forces rapid dissociaLon and facilitates stable complex Results of Hinkle and Chamberlain’s experiment # stable % # loose total E/DNA % Ratio stable complexes loose complexes 8/1 100 8 0 0 8 13/1 60 8 40 5 13 26/1 30 8 70 18 26 Shapes of E.coli RNAP core (a) holoenzyme (b) Model for RNAP interaction with promoter DNA Temporal control of Transcription in phage SPO-1 infected B. subtilis T7 RNA Polymerase. Lambda Life Cycle. Lambda Lambda Genome Plaques are turbid Lytic Life Cycle. N anti-terminates transcription. Protein complexes involved in N-mediated anti-termination. LyLc Growth Summary PL and PR are used by RNAP no cI present Cro and N are made N anL-‐terminates tx at tL and tR1 Polymerase transcribes cIII and recombinaLon genes (L) • Polymerase transcribes cII and DNA replicaLon genes (R) • Cro binds to OR and OL blocking the PRM • If cII levels are low then lyLc growth conLnues because the PRE is not acLvated • • • • Lambda Cro vs cI • Cro and cI are DNA binding proteins that bind to the operator sequences OR and OL • There are three binding sites for each protein in the operator • Cro and cI bind with opposite aﬃniLes to these three sites • cI binds ﬁrst to site 1, then 2 and at high concentraLons site 3 • Cro binds to site 3 ﬁrst, then site 2 followed by site 1 • cI binds to sites adjacent to its own promoter PRM acLvaLng its own transcripLon • Cro binds to the PRM and prevents transcripLon from this promoter The Battle between cro and cI. Q-mediated anti-termination of the late genes. Establishing Lysogeny If cII levels are high then lysogenic growth will proceed cII acLvates PRE cII acLvates PI cII acLvates PanLQ ResulLng in the synthesis of cI and the turn oﬀ of PR eliminaLng cro synthesis • cI binds to the OL and OR prevenLng further PL and PR transcripLon • cI acLvates its own promoter PRM and lysogeny is established • • • • • Lambda Establishing Lysogeny. Control Region Maintaining lysogeny. The Life-‐Cycle Decision • The levels of cII and cIII are criLcal they sense the ‘health’ of the cell • Healthy rapidly growing cells have high levels of proteases which degrade cIII and cII • cIII tries to block the proteases from cleaving cII • Hﬂ (high frequency lysogen) is a bacterial gene that greatly inﬂuences this decision • When hﬂ is absent or mutated lysogeny is highly favored as this protease cleaves cIII • When cells are starved and not dividing protease levels are low and cII and cIII levels are stabilized thus favoring lysogenic growth Lambda IntegraLon • Once cII levels are established cII acLvates the PI promoter allowing the synthesis of int • PI is located in the xis gene so only int is made and integraLon results Role of PI • TranscripLon from the circular lambda DNA from PL generates a transcript that contains the ‘b region” • These b region in the transcript prevents translaLon of the int gene and only the xis protein is made and the phage cannot integrate • Thus cII acLvaLon of PI blocks xis and allows int to be made and integraLon to occur Control Region cII; Pre and PanLQ Lambda Lambda inducLon • DNA damage results in the acLvaLon of the SOS system in bacteria and the synthesis of recA • The recA protein causes cI to cleave and dissociate from DNA • When OR and OL no longer have cI bound PR and PL become acLve and lyLc gene expression proceeds Inducing the Lambda prophage. Excision of Lambda DNA • IntegraLon of lambda DNA into the genome results in the physical separaLon of the b-‐ region from int and xis • Under these circumstances the synthesis of both xis and int occur from PL • When both int and xis are present excision will occur and the prophage is excised from the genome Why are plaques turbid?
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