Transcript Microbial Genetics - University of Montana

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 plasmid-
mediated gene transfer
– Reading
• Snyder and Champness, Chapter 5