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Transcript
Figure 2.6 Part 1 Joining DNA after a single enzyme has cut it Vector DNA Donor DNA Vector plus one or more donor fragments EcoRI AATTC G AATTC G G CTTAA G CTTAA Reclosed vector Circularized donor Copyright 2006 by E.A. Birge Figure 2.6 Part 2 Joining DNA after two enzymes have cut it Vector DNA Donor DNA Vector plus one or more donor fragments Keep large fragment EcoRI PstI CTGCA G Discard G CTTAA small fragment G G CTTAA ACGTC AATTC G G ACGTC Only one possible structure can form and be joined by DNA ligase Copyright 2006 by E.A. Birge Figure 4.7 RNA Polymerase Binds all the time lacZ lacY lacA lacI PI Transcripts Binds only when CRP and cAMP are present P3 P2 O3 P1 O1 O2 Low efficiency promoters Translation gives repressor monomer O1 tL High efficiency promoter Both of these mRNA moleculestetramer are Repressor may translatable to give bind to operator sites, b-galactosidase, forming a loop in the DNA galactoside permease, that prevents RNA O2 and thiogalactoside polymerase transacetylase binding lacY Repressor monomer may lacI P1 bind to allolactose, the true inducer Copyright 2006 by E.A. Birge Figure 4.8 RNA polymerase binds here in the absence of cAMP and CRP RNA polymerase binds here when repressor absent and cAMP and CRP bound galM galK galE galE galT galT galK galK galU galUgalS galS galR galR PR PRtR OtRE P2OP E 1PO 2 IP1 OI Translation tL PUtL PtUU PtSU PtSS tS Arrows indicate mRNA transcripts Translation Repressor monomer Alternate repressor Inside of loop works best at PS but also binds OI and OE Repressor binds to galactose and leaves promoter open P2 OE P1 OI galE Repressor dimer bridges OE and OI, blocking transcription from P1 but not P2 Copyright 2006 by E.A. Birge Fig. 4.10 Regions DNA duplex that can in form the attenuator stems and region loops 1 2 3 4 RNA polymerase pause site. Ribosome near the end of Approach of ribosome starts the leader peptide Polymerase moving again. No translation Ribosome paused at tryptophan codons 2 2 1 3 2 4 3 1 4 1 Attenuator loop forms, transcription stops Antiterminator loop forms, transcription continues Copyright 2006 by E.A. Birge 3 4 Fig. trpE trpD trpC trpF trpB trpA hisH tyrA argE p p 4.11 Leader region 1 50 100 Excess tryptophan 150 Antiterminator loop Limiting tryptophan 1 50 Trap wheel binds to DNA 150 100 100 150 Potential terminator loop Terminator loop 50 1 trp operon has two promoters, only 1st is attenuated mtrB encodes trap protein mtrA mtrB p Tryptophan Copyright 2006 by E.A.2002 Birge Copyright by E. A. Birge Uncharged tRNA stabilizes antitermination loop I UA G G CC C G GU GCCA Tryptophan in short supply, tRNA uncharged Fig. 4.12 Antiacceptor loop Potential attenuator stem Codon equivalent Tryptophan prevents binding to antiacceptor loop II III UA G C C G Tryptophan in excess, tRNA is charged Copyright 2002 by E. A. Birge Copyright 2006 by E.A. Birge GCCATrp UGGC Terminator loop forms Figure 5.4 Gap Nick Replicate Cut strand D-loop forms Replicate Branch migration to the right Copyright 2006 by E.A. Birge Isomerization (can branch migrate in either direction) Figure 5.10 Chi site + As this strand emerges, other strand binds to it + 3’ or Eventually Exo V is site RecBCD Now have a Properly oriented Chi marching along DNA single triggers endonucleolytic cut strand Final Products that can bind RecA and Nucleoprotein filament form a D-loop D-loop Holiday Structure synapses with new DNA Copyright 2006 by E.A. Birge Figure 6.6 Wild type phage Mutant phage with same phenotype Mutant phage E. coli host cell Trans test (same cistron) Trans test (different cistrons) Cis test a B A b No progeny can result because a and b affect the same enzyme a b A B Both enzyme A & B are functional, lysis occurs Copyright 2006 by E.A. Birge a B A b Complementation DNA protrudes through membrane Primer Figure 7.9 RF IV Infecting virus Coat protein accumulates in membrane Complementary strand synthesis Gene V New virus protein replaces SSB Displaced strand Unpackaged stabilized by SSB DNA Copyright 2006 by E.A. Birge RF II (nicked by protein A) Rolling Circle Displaced DNA is coated with SSB Protein A can rebind and nick DNA duplex Figure 8.2 Box B loop RNA Nutpolymerase has two binds and starts subsites, A and transcription B nut P (Early promoter) NusA protein required for termination NusB binds to Box A and S10 Box A binding site Delayed early Genes located here l DNA T (Transcription terminator) Ribosomal protein Lambda N S10 binds to RNA protein binds pol and NusB to NusA and Box B NusG normally causes termination. Displaced by NusB Copyright 2006 by E.A. Birge Figure 8.4, lytic response Possible transcripts PRM PL PRE PR att int xis red cIII N OL rex cI OR cro cII O P Q CR OL1 CR OL2 PL Repressor binds to 1, then 2, then 3 CR OL3 CR OR3 PRM CR OR2 CR OR1 PR Repressor Left and right Cro binds to 3, All transcripts maintenance transcripts then 2, then 1 turned off promoter turned off turned off on Copyright 2006 by E.A. Birge Figure 8.4a, Temperate response PL PRM (L1M transcript) PRE (L1E transcript) att int xis red cIII N OL rex cI OR cro cII O P Q R OL1 R OL2 R OL3 R OR3 Cro PRM binds Left and right Repressor binds to All transcripts to 3, then 2, transcripts 1, then 2, then 3 except repressor then 1 turned off turned off PL Copyright 2006 by E.A. Birge R OR2 R OR1 PR Repressor maintenance promoter turned on Figure 8.4b l Repressor Binding Adjacent dimers form tetramers OL1 OL2 OL3 Repressor dimers Binding of final tetramer blocks last active promoter OR3 OR2 OR1 Tetramers then form an octamer, looping the DNA OL3 OL2 OL1 PRM OR3 OR2 OR1 The issue that remains is how PRE turns on Copyright 2006 by E.A. Birge Turning on PRE PInt PRE att int xis red cIII N OL rex cI OR cro cII O P Q cIII protein antagonizes ftsH protein Critical Genes are cII and cIII cII protein binds to –35 region of two promoters, has same effect as CRP on lac promoter Host protein ftsH cleaves cII, keeps PRE turned off Therefore, if cII or cIII proteins are mutated, very difficult to turn on these promoters. If they do turn on, get a normal lysogen. Copyright 2006 by E.A. Birge Figure 10.1, DNA Uptake Input DNA Pseudopilin subunits Outer membrane Peptidoglycan NucA Pilin subunits N N Cell Membrane ATPase Single-strand fragment Copyright 2006 by E.A. Birge Fig. 10.2 DNA Entry into Hemophilus Donor is cleaved DonorDonor DNA DNADNA Donor DNA Donor DNA is internalized DNA Receptor Protein Outer membrane Cytoplasmic membrane One strand is degraded, the other is translocated to cytoplasm Copyright 2006 by E.A. Birge Fig. 11.2 Interrupted Mating If you extrapolate from late value, intercept doesn’t make sense Need to extrapolate from earliest time points Copyright 2006 by E.A. Birge Fig. 11.8 Transfer DNA Replication Transfer to light, radioactive medium Transfer to light, nonradioactive medium Replicating Hfr Note that all DNA labeled with By chance, someDNA labelis either 13 and 15N (heavy the or If C new round of will be at oriT,heavy:light isotopes) of Next round of replication begins plasmid originheavy:heavy replication replication begins at plasmid oriT, get radioactive light:light DNA early At this point, the At this point, all radioactive DNA DNA is heavy: becomes light:light light Copyright 2006 by E.A. Birge Fig. 12.6 Conjugal Plasmid Interactions R100 Plasmid finO traJ finP Naturally defective F Plasmid protein in F, therefore finO transfer always is traJ finP efficient FinO works in trans so R100 reduces transfer by F as well as and FinP levels build up, and conjugal itself ability decreases. Transfer is very efficient right after a previous transfer. Required for The combination of FinO and conjugal functions FinP proteins inhibits traJ function to be expressed When R100 arrives in a cell, FinO Copyright 2006 by E.A. Birge Fig. 13.1 Plasmid R1Copy Number Origin of Promoter 2 Copy number control region replication CopA RNA Prevents translation of tap Promoter 1 tap copB CopT RNA Translation of tap allows translation of repA CopB inhibits promoter 2 activity RepA stimulates replication initiation Copyright 2006 by E.A. Birge CopB RNA Translation gives CopB protein repA Fig. 13.1 pIP501Copy Number Origin of When bound, 2 causes replication RNA IIIPromoter attenuation of RepR mRNA Promoter 1 copR repR CopR RNA Translation gives CopR protein RepR RNA CopB inhibits promoter 2 activity and stimulates Promoter III Copyright 2006 by E.A. Birge RepR stimulates replication initiation F or P1 Plasmid Partitioning ADP plus sopA protein inhibits promoter (autoregulation) ADP ATP plus proteins causes partitioning of plasmid at ¼ and ¾ ATP of distance to pole of cell F sopA sopB sopC P1 parA parB parS Copyright 2006 by E.A. Birge Fig. 14.2 Nitrogen Regulation (Global Regulatory Network) NtrC (inactive) NtrB (kinase phosphatase) GlnD + glutamine GlnB-UMP NtrC-P GlnD + 2-oxoglutarate NtrC-P NtrC-P NtrC-P Need two glnA ntrBC nifL nifA glnK amtB Now focus only factors to on regulatory s54 s54 s54 activate proteins NifA GlnK transcription, Activates NifL (interferes activator must Promoter promoter Inhibits nifHDK with NifL) bind to upstream NifA 54 enhancer s function GlnB Nitrogenfixation fixation Nitrogen turned occursoff occurs Copyright 2006 by E.A. Birge Fig. 14.4 Sigma Factor Production Sigma A and phosphorylated Spo0A trigger this promoter Transcription and translation sigE Prosigma E Sigma E activates this promoter spoIIID Transcription and translation Sigma E Sigma E Activates newly spliced gene SpoIIID protein Activation spoIIIC spoIVCA spoIVCB spoIIIC spoIVCB Prosigma K DNAActivated sequencespoIVCA analysis shows is a that the DNA coding for Sigma K Two components of sigma recombinase K is actually thatsplit catalyzes by a gene called spoIVCA spoIVCA turns on late genes in Sigma K excision of its own gene is discarded mother cell, turns off sigE Copyright 2006 by E.A. Birge Fig. 15.2 Gin-catalyzed Inversion –1 This is a ribbon diagram with blue on one side of the ribbon and brown on the +1/2 +1/2 other. The circular molecule is folded so that the enhancer (red) passes between the two vertical strands. The large arrows are the gix +1 sites. +1 –1 Before recombination After recombination Copyright 2006 by E.A. Birge Copyright 2002 by E. A. Birge Fig. 15.4 Tn10 Transposition IS10L Bent target DNA TetR IS10R Nearly precise excision or Precise excision Copyright 2006 by E.A. Birge Fig 15. 4A Phage Mu Transposition Bacterial DNA L1 L2 L3 E R3 R2 R1 attL attR Mu Prophage B B Gene A product Transposome assembles is a transposase A A A A IHF IHF IHF bends Mu DNA Target DNA Copyright 2006 by E.A. Birge ATP ADP PhageAMu B Protein causes protein binds hydrolysis of to followed target ATP bysequence release of B protein Fig 15.5B Molecular Rearrangements This is strand transfer complex Single Mu prophage strand nicks in in host prophage DNA Replication begins, target sequence duplicated at each end Offset cuts Target DNA Target DNA separates 5 bp apart Copyright 2006 by E.A. Birge Fig. 15.4B After Replication Finishes ButThis the regions molecule containing is lined upthefortwo recombination prophages are Whenreally you straighten Aout crossover circular occurs DNA, you get part of within one big thethe cointegrate prophages molecule within the prophages Copyright 2006 by E.A. Birge After Recombination Finishes These regions are also duplicated, but that event Noteoccurred that this during was essentially a resolvase reaction the previous transposition Original DNA molecule restored, although prophage is actually a recombinant Prophage is flanked by short duplication Target DNA molecule is more complex Copyright 2006 by E.A. Birge Copyright 2002 by E. A. Birge Ch 17 Operon Organization bgl operon Phospho-bglucosidase bglB Pts Enzyme II bglF antiterminator bglG transcript Degrades sugar Transports sugar Copyright 2006 by E.A. Birge or phosphorylates (inactivates) antiterminator protein Defective promoter can be activated by bglO or leuO Ch 17 Tree Building • The sequence differences must be informative • Simple example Progenitor Sequence Descendant 1 Descendant 2 Descendant 3 AAGGCCTT AAGGCCTT AAGGCCTT ATGGGCTT ATGGGCTT ATCGGCTT ATCGGCTT ATCGGCTT ATCGCCTT ATCGCCTT ATCGCCTT Overall, descendant 2 has two differences The indicated are different from from the progenitor and therefore most Descendant 1descendant matches the progenitor 2 is and 3 do not, This time 3 bases matches thebut progenitor but distant it. but 1 closely and 2 are equally distant. 1 is more closely to not progenitor than 2 or 3, which 1so and 2 do not, so 3from isrelated more related to the progenitor progenitor from each other There is enough information arenot related to each other to sort so theyout are uninformative 1 and 3 Copyright 2006 by E.A. Birge
