1. Microbial Genetics MICB404, Spring 2008 Lecture #13 Biology of plasmids: II. Modes of replication

2. Announcements -Summary due today. -Review Wednesday -Exam Friday

3. Regulation of copy number by antisense RNA Plasmid R1Plasmid ColE1

4. 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

5. 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

6. ColE1 plasmids

7. R1 copy number control RepA protein Plasmid-specific replicative helicase

8. 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

9. R1 copy number control repA gene transcribed from 2 promoters on plasmid 1) p repA : RepA mRNA located in copB repressed by CopB protein 2) p copB : CopB-RepA polycistronic mRNA Regulatory antisense RNA, CopA, transcribed from p copA –constitutive

10. R1 copy number control Transcription from p repA only occurs immediately after transformation –RepA then drives replication until copy number reached –expression of copB results in repression of RepA expression from p repA –repA can now only be transcribed from the p cobB promotor

11. 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

12. 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

13. 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

14. Iteron plasmids ori contains repA gene –Sole plasmid-encoded protein required for replication –3 iteron sequences, R1, R2, & R3

15. 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

16. Iteron plasmids II. Coupling –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]

17. Iteron plasmids

18. 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.

19. Eukaryote 2μ Inverted repeats Partitioning into daughter cells During mitosis and meiosis “Flip protein” Site-directed recombinase Proteins repressing expression of FLP (constitutive)

20. Plasmid 2μ

21. Plasmid maintenance Curing –Loss of all plasmids from cell after cytokinesis –Prevented by plasmid addiction multimer resolution partitioning

22. 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

23. Restriction endonuclease toxicity CH3 | GTATGCTCAC CATACGAGTG Methylase CH3 endonuclease X Plasmid curing GTATGCTCAC CATACGAGTG endonuclease Methylase

24. 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

25. 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

26. 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

27. R1 ParM-based segregation ATP hydrolysis is proposed to induce a structural change in ParM. ParR is released and reassociates with ParM-ATP. ParR N-terminal binds specifically to parC while the C-terminal interacts with ParM-ATP.

28. R1 ParM mediated partitioning ParM foci ParR bound to parC site DNA rep. apparatus Plasmid tethered to pole via ParM and ParR/parC Movement to mid-cell, replication, ParR binding Rapid movement to cell poles Dissociation of filament

29. 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

30. 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

31. 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

32. 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

33. Monday’s lecture: –Conjugation: Mechanisms of plasmid- mediated gene transfer –Reading Snyder and Champness, Chapter 5