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Microbial Genetics MICB404, Spring 2008 Lecture #13 Biology of plasmids: II. Modes of replication • Announcements -Summary due today. -Review Wednesday -Exam Friday Regulation of copy number by antisense RNA Plasmid ColE1 Plasmid R1 ColE1 plasmids • Copy number regulated by siRNA (RNA I) inhibiting primer RNA (pRNA, RNA II) availability – pRNA: 550 nt • RNA Pol product • processed by RNase H • anti-sense RNA (RNA I) transcribed from opposite strands of the pRNA locus – RNA I: 108 nt – complementary to 5’ end of RNA II ColE1 plasmids • RNA I:RNA II duplex forms – step 1, small number of base pairs (“kissing complex”); rate-limiting – step 2, full-length duplex formed (“hug”) – prevents RNase H processing of RNA II and formation of RNA:DNA primer complex • RNA I transcribed from constitutive promoter – Increasing plasmid copy number yields more RNA I, thus increasing inhibition of replication ColE1 plasmids R1 copy number control RepA protein • Plasmid-specific replicative helicase R1 copy number control • RepA required to initiate plasmid replication • therefore control of Rep protein concentration will control copy number • Antisense RNA inhibits expression of Rep protein – Plasmid-encoded R1 copy number control • repA gene transcribed from 2 promoters on plasmid 1) prepA: RepA mRNA • located in copB • repressed by CopB protein 2) pcopB: CopB-RepA polycistronic mRNA • Regulatory antisense RNA, CopA, transcribed from pcopA – constitutive R1 copy number control • Transcription from prepA only occurs immediately after transformation – RepA then drives replication until copy number reached – expression of copB results in repression of RepA expression from prepA – repA can now only be transcribed from the pcobB promotor R1 copy number control • CopA RNA binds to CopB-RepA mRNA – double-stranded RNA forms over region spanning 5’ end of RepA ORF • upstream of repA is a short leader peptide ORF • translationally coupled with RepA R1 copy number control • CopA:RepA dsRNA cleaved by RNase III in leader peptide ORF – interferes with RepA translation – more plasmid CopA RNA – more CopA RNA less RepA protein – limiting RepA protein no plasmid replication Iteron plasmids: copy number control • Some plasmids contain iterated (repeated) sequences in oriV – e.g. pSC101, F, RK2 • pSC101 first plasmid used for cloning recombinant DNA: 1973, frog rRNA genes cloned into EcoRI site – 17 to 22 bp – 3 to 7 copies per plasmid Iteron plasmids • ori contains repA gene – Sole plasmid-encoded protein required for replication – 3 iteron sequences, R1, R2, & R3 Iteron plasmids • Two-part copy number regulation I. RepA protein multi-functional – Required for replication – Represses transcription of repA gene • Transcriptional auto-regulation • Increasing plasmid copy number increasing RepA protein increasing repression of repA expression II. Coupling Iteron plasmids – RepA protein binds to iteron sequences • Low plasmid concentrations – bimolecular interaction – replication activated • High plasmid concentrations – multimolecular interaction – “handcuffed” or “coupled” plasmids prevented from replication – Results in replication control according to [RepA] and [plasmid] Iteron plasmids Eukaryote plasmid 2μ • Typically, 50 to 100 copies per cell • Replication initiated only once per cell cycle • Bidirectional and rolling circle replication • Regulation of recombinase expression. Eukaryote 2μ Proteins repressing expression of FLP (constitutive) Inverted repeats “Flip protein” Site-directed recombinase Partitioning into daughter cells During mitosis and meiosis Plasmid 2μ Plasmid maintenance • Curing – Loss of all plasmids from cell after cytokinesis – Prevented by • plasmid addiction • multimer resolution • partitioning Plasmid addiction • Plasmid-encoded factor that kills cells cured of plasmid – plasmid also encodes “antidotes” to toxic protein – upon curing, antidotes are lost and cell is killed by toxic protein – Toxicity • aberrant DNA gyrase • disrupt membrane potential • etc Restriction endonuclease toxicity Methylase CH3 | GTATGCTCAC CATACGAGTG Plasmid curing Methylase GTATGCTCAC CATACGAGTG endonuclease endonuclease Multimer resolution • Plasmid replication can result in formation of dimers & multimers – Result in increased curing • Prevented by site-specific recombination – Resolve multimers into monomers • Plasmid or chromosome encoded Multimer resolution • Site-specific recombinase – XerC and XerD proteins • encoded by chromosomal genes – Promote recombination between cer sites on plasmid – Irreversible • recombination does not occur between cer sites on monomers – Cytokinesis delayed until recombination complete Partitioning • System that segregates plasmids into each daughter cell – Probability of segregation – 50:50 chance per plasmid; either cell: 2.(1/2)2n n = copy number = 1/128 R1 ParM-based segregation ParR N-terminal binds specifically to parC while the C-terminal interacts with ParM-ATP. ATP hydrolysis is proposed to induce a structural change in ParM. ParR is released and reassociates with ParM-ATP. R1 ParM mediated partitioning Plasmid tethered to pole via ParM and ParR/parC Movement to mid-cell, replication, ParR binding Rapid movement to cell poles Dissociation of filament ParM foci ParR bound to parC site DNA rep. apparatus Incompatibility • Some plasmids are incompatible in the same cell – mutual interference in replication or partitioning • Inc group defines plasmids which are unable to coexist in same cell Compatible plasmids Incompatibility • Replication control – Two plasmids of same Inc group and same replication control will share copy number between them • unequal replication will result in declining proportion of one plasmid • eventually that plasmid is cured from cell – Particularly with stringent plasmids • low copy number Incompatibility • Partitioning – Two plasmids using same par system will be incompatible – They will be partitioned into one or the other daughter cell at random • one daughter receives only plasmid X, other receives on plasmid Y – Stringent plasmids Incompatibility • Measurement of plasmid curing – Incompatibility test: two plasmids with different antibiotic resistance genes • Higher rate of curing for 2 plasmids together indicates they are of same Inc group Maintaining antibiotic selection for both plasmids can overcome loss of plasmids from same Inc group • Monday’s lecture: – Conjugation: Mechanisms of plasmidmediated gene transfer – Reading • Snyder and Champness, Chapter 5