Plasmids Flashcards
what is plasmid DNA?
extrachromosomal DNA, found in all types bacteria, sometimes more than 1 type of plasmid in 1 cell
what do plasmids typically look like?
-usually covalently closed, circular, dsDNA
-vary in size, sometimes difficult to determine which DNA molecules is plasmid or chromosome
How can you maybe tell plasmid apart from chr?
cure cell of “plasmid” by removing it. If the cell lives, it is a plasmid. If the cell dies, it is the chromosome
what type of proteins and RNA do plasmids typically encode?
benefit bacterium in a specific environment (ex. antibiotic resistance)
what is the F plasmid?
fertility plasmid (make pilus)
what are IS genes?
Insertion sequences; allow for insertion
what is the oriT?
origin of transfer; allows for conjugation
what is oriV?
vegetative growth; the origin for replication
what proteins do tra genes encode?
proteins involved in plasmid transfer
F+ cell have what?
has F plasmid
F- cell is what?
is the recipient, doesn’t have F plasmid
plasmids were named initially named after what characteristic?
functions encoded (ex. R plasmids = encode antibiotic resistance)
now, plasmid naming is standardized. how?
pTR1
(plasmid)(initials of creator)(version)
are plasmids negative or positive supercoiled?
negative
Supercoil stress of plasmid is relieved by what? This characteristic is used to facilitate reactions like replication and transcription
-protein binding
nicked plasmids are what?
relaxed, no supercoiling
small size of plasmids allows for its isolation among bacterial chromosome and other macromolecules. What technique is used?
selective precipitation
What are the steps for plasmid purification?
-bacteria are lysed with detergent and base
-chromosome & cellular constituents are preferentially precipitated with concentrated salt solution
-soluble plasmid DNA is bound to a column, washed, & eluted
what is a replicon?
-dna that can replicate independently (ex. plasmids)
plasmids encode only one protein required for initiation of replication. Where is this gene?
Only this small region of plasmid is required for replication. The rest required are borrowed from host machinery. What gene is it? rep gene
near oriV
rep gene
What is theta replication?
-RNA primer initiates DNAP replication in one or both (bidirectional) directions around plasmid
Theta replication is used by what plasmids/cells?
- ColE1, RK2, F, P1 plasmid
-also used by bacterial chromosome (but not called theta replication) & larger plasmids
What is Rolling Circle replication?
-ds circular plasmid replicates to generate ssDNA
-complementary strand synthesized on new ssDNA to generate new plasmid
Rolling Circle replication is used by what systems?
-some phages
-during bacterial conjugation
What are the steps of Rolling Circle replication?
1)Rep protein binds ds oriV (DSO) @ palindrome and causes formation of hairpin cruciform structure. Rep then nicks DNA (binds Tyr to 5-PO4)
2) Then DNA Pol III extends 3-OH
3) Rep transfers 5–PO4 to 3’-OH to create circular ssDNA (original DNA)
4) lagging strand synthesis occurs on displaced strand
5) host RNAP makes primer at ss oriV (SSO)
6) DNA Pol III replicates around ssDNA circle
7) DNA Pol I removes RNA primer, replaces with DNA, ligase seals
rollin circle replication can also produce this DNA structure: the original parental DNA sequence is repeated many times
long concatemers
What does the host ranges of plasmids describe?
the types of bacteria a plasmid can replicate in
What is a Narrow host range & what type of plasmids have narrow range?
-replicate only in E. coli & closely related microbes
-ColE1 plasmids -> pBR322, pET, pUC
What is a broad host range & what type of plasmids have broad range?
-replicate in many Gram negatives, some Gram
positives!
-RK2/RSF1010 derivatives -> pBBR1MCS
broad host range plasmids are broad host for these two reasons?
-encode all proteins required for replication, not dependent on host
-gene expression sequences recognized by wide variety of machinery (ie. promoters, translation initiation sites)
How can you determine the host range of plasmids? (2)
-Way to introduce plasmid into different hosts
-Selectable marker (some phenotype encoded by plasmid that indicates presence in bacterium - ex. antibiotic resistance)
What is the copy number of plasmid?
- characteristic number of copies per cell (highly controlled)
what are relaxed plasmids?
- plasmids with high copy numbers that control the initiation of replication when plasmid reaches high numbers
what are stringent plasmids?
- plasmids with low copy numbers that tightly control the initiation of replication (maybe only one copy per cell)
what is plasmid incompatibility?
- when two plasmids cannot coexist stably in same cell
plasmid incompatibility occurs for two reasons? what are they?
-plasmids use the same replication mechanism
-plasmids use the same partitioning functions
What can happen if two plasmids use same replication mechanism?
i. Replication control mechanisms do not recognize plasmids as different
ii. Either plasmid selected randomly for replication
iii. Eventually one will be lost, by chance
What can happen if two plasmids use same partitioning functions?
i. Partitioning mechanisms do not recognize plasmids as different
ii. Plasmids will be partitioned unevenly to daughter cells
iii. Eventually one will be lost
plasmids that cannot be maintained together in same cell are said to belong to ______
same incompatibility/inc group
How can you determine the inc group of a plamid?
you can measure whether is it cured from strains containing plasmids of known inc group
What are 3 mechanisms used to control replication of plasmids? plasmid type associated with them
I. interference with primer for DNA replication by small RNA (sRNA): ColE1, pUC,
II. control of Rep protein copy number with complementary RNA: R1
III. Rep protein autoregulation & plasmid coupling via ITERONS: pSC101
How does interference with DNA replication by sRNA control plasmid replication? (cloning plasmids: ColE1, pUC)
-RNA II normally binds at ori and is processed to form primer for replication
-As plasmid replicates, complementary sRNA RNA I accumulates and Interacts with RNAII using Rop dimer and prevents priming of replication to control copy number
How does control of Rep protein copy number with complementary RNA control plasmid replication (Plasmid R1)
-Most plasmids needs a Rep protein that binds to specific oriV to help separate DNA strands prior to replication
-Plasmid encoded sRNA accumulates as plasmid copy # increases
- sRNA inhibits translation of Rep, thereby keeping plasmid number at a certain level.
How does plasmid replication shutdown happen with repA? What is different in Gm+ cells?
-plasmid encodes CopB repressor protein that shuts off repA gene
-CopA RNA made by plasmids & binds repA transcript to inhibit translation
-In Gm+ cells, sRNA stops transcription of rep gene.
How does Rep protein auto-regulation & plasmid coupling via iterons control plasmid replication (pSC101)
-When Rep protein accumulates, feedback inhibits its own promoter to shut off expression
-At high levels of Rep & replicated plasmid, Rep proteins bound to iterons in oriV interact to couple plasmids & prevent replication
-aka handcuffing
Plasmids Partitioning carried out by what? These process is similar to that of bacterial chromosome segregation
-Par functions/Par proteins
-bacterial chromosome
for high-copy number plasmids, _______ partitioning occurs. what about low copy plasmids?
-random
-plasmid replication is coordinated with chromosome replication so cell division partitions plasmid into daughter cells
2 kinds of partitioning
1) R1 type
2) P1/F type
What is the R1 type (search & capture) partitioning?
-ParR protein binds centromere-like parC site on plasmid
-ParR bind ParM
-ATP-bound ParM polymerizes, much like actin, under ParR:parC complex
-ParM-ATP polymerization drives replicated plasmids
apart
-ATP hydrolysis causes ParM to depolymerize
What is P1/F type partitioning?
-ParA-ATP polymerizes at random places on nucleoid (chromo DNA)
-ParB binds to parS site on plasmid DNA
-ParB-parS attracted to ParA-ATP
-ParB-parS stimulates ATP hydrolysis & depolymerization of ParA
-ParB-parS (and plasmid) pulled across chromosome as ParA depolymerizes
What is the centromere in partitioning systems: Plasmid R1 vs Plasmid P1/F
R1: parC
P1/F: parS
What is the DNA binding protein in partitioning systems: Plasmid R1 vs Plasmid P1/F
R1: ParR
P1/F: ParB
What is the actin-like protein & where does it act in partitioning systems: Plasmid R1 vs Plasmid P1/F
R1: ParM-ATP (at midpoint)
P1/F: ParA-ATP (on nucleoid)
What is the movement in partitioning systems: Plasmid R1 vs Plasmid P1/F
R1: pushes DNA apart (filament polymerizes to move DNA)
P1/F: pulls DNA apart(filament depolymerizes to move DNA)
