Plasmids Flashcards
What are Plasmids?
small, circular, extra-chromosomal DNA which replicates independently of host chromosomal DNA
Are all plasmids circular?
No, they are not.
R-factors in plasmids give bacteria what?
Antibiotic resistance
How many proteins can plasmids encode for?
Depending on their size, they can encode either a few or hundreds of different proteins
Do plasmids give large amounts of advantages to bacteria?
No, they give bacteria selective advantage only in certain conditions
When purifying plasmids, after hours of ultracentrifugation, where in the tube would you find the plasma DNA?
Due to a formation of a gradient, the plasmid would be the lower band showing
The EtBr bound to DNA makes it ___________ in salt solution made with heavy atoms such as CsCL.
less dense
What are the 3 properties of plasmids?
- Replicon (DNA molecules that can replicate autonomously in the cells
- Plasmids has one origin of replication (oriV)
- The ori region determines the mech. of replication
What are the two types of plasmid replication?
- Theta replication
2. Rolling circle
What is Theta replication?
Replication that begins by opening the two strands of DNA at the ori region, creating a structure that looks like the Greek letter “Theta” (circle with line through it)
What is unidirectional replication?
Replication that terminates when the replication fork gets back to the origin
What is bidirectional replication?
Replication that terminates when the replication fork meets somewhere on the DNA molecule opposite the origin
Rolling circle plasmid replication for this mechanism are named ________.
RC plasmid
There are two stages to rolling circle plasmid replication. What are they?
- Replicated and form double and single strand circular plasmid
- The complementary strand is synthesized on the single-stranded DNA to make another double-stranded DNA
What kind of protein is encoded by the plasmid?
Rep protein
What are the functions of the Ori regions?
- Host range
2. Regulation of copy number
When dealing with the Host range of functions, what are the ranges seen as functions of the Ori regions?
- Narrow host range
- Broad host range
- Incompatibility
- Plasmid replication control mechanism
What regulation of copy number functions of the Ori region is?
Relaxed plasmid, molecules which inhibit a plasmid replication when the number reach certain level (mechanism present in high copy plasmids
Plasmid incompatibility is when plasmids ____________, and one or the other will be lost at a higher than normal rate when the cells divide
can not coexist
What is Incompatibility group (Inc group)?
It is the plasmid members of the same Inc group that are not able to coexist
Why can’t plasmids members of the same Inc group are not able to coexist?
Because they share the same replication control mechanism and/or the same partitioning (par) functions
When dealing with plasmid replication control mechanism, what type of plasmid is regulated by a ctrRNA and Protein?
Cole1-derived plasmid
R1 and ColB1-P9 plasmid does what in bacteria?
Regulation of Rep protein by antisense RNA
How are Iteron plasmids regulated?
Regulation by Coupling
What type of plasmid are ColE1-derived plasmid?
Endogenous plasmid
ColE1-derived plasmid makes _______ protein
Immunity
Many plasmid cloning vector are derived from what type of plasmid?
ColE1-derived plasmid
ColE1-derived plasmid replication is regulated by?
RNA I (small RNA encoded by plasmid)
In ColE1-derived plasmid, RNA I interfere with the processing of RNA II by?
forming a double strand RNA.
RNA I and RNA II are ___________.
complementary
What forms the primer for plasmid replication in regards to ColE1-derived plasmid?
RNA II
Do ColE1-derived plasmid require encoded proteins to initiate DNA replication at their oriV?
No, they do not require encoded protein.
Imm stands for?
Immunity protein
Kil stands for?
lyses protein
Inc stands for?
RNAI (ctRNA)
rnall stands for?
replication primer
oriV stands for?
origin
Rop does what?
regulates copy number
Bom stands for?
origin of transfer
Mob stands for?
transfer protein
Cer does what?
maintains monomers
What does Exc do?
excludes incoming plasmids
What does Cea mean?
Colicin toxin
What are the two mechanisms of regulation?
- Stop replication
2. Initiate replication
How does stop replication work? (Mech. of Regulation)
Presences of RNA I –> Pairing of RNA I - RNA II (short sequence - kissing complex)–>Rop protein stabilize the kissing complex–> RNA I-RNA II form a double stranded RNA–> Prevent formation of secondary structure of RNA II required to hybridize DNA
How does initiate replication work? ( Mech. of Regulation)
Absences of RNA I–> RNA II form a hybrid RNA-DNA at ori–> RNA H cleaved RNA II–> 3’-OH is release serve as primer for first replication
More plasmid –> __________ –> stops replication
more Rop + RNA I
Less plasmid –> ____________ –> allows replication
les Rop + RNA I
Rop + RNA I —> ?
no plasmid replication
RNA II –>?
plasmid replication
T or F: ColE1 plasmid do not require a plasmid-encode protein to initiate replication at their oriV, only an RNA primer synthesized from the plasmid
True
What type of plasmid-encoded protein do most plasmids require?
Rep
What does “Rep” do?
It separates the strand of DNA at oriV region
Is “Rep” specific?
It is very specific, only binds to oriV from the same plasmid –> recognize specific sequences
T or F: The synthesis of Rep fully controls the copy number of the plasmid
False: it only partially controls the number of plasmid copies
What is done to inhibit the synthesis of Rep protein?
An antisense RNA (called CopA)
How does antisense RNA work?
Antisense RNA forms a hybrid
Hybrid prevents translation of what?
Rep A
What does RNase III target?
The Hybrid
T or F: RNA III is a chromosomal enzyme
True
When does replication occur?
Replication occurs after plasmid enters the cell
What is “iteron sequence”?
It is an oriV region that contains several repeats of a certain set of DNA bases
The interon sequence has ___ to ___ bp long and exist from ___ to ___ copies in this region.
17 to 22; 3 to 7
What does repA gene encode for?
RepA protein
What makes RepA protein so important?
it is the only plasmid-encode protein required for replication
The host chromosome encodes the other proteins that bind to this region to allow initiation of the replication. What are they?
DnaA, DnaB, DnaC, and DnaG
RNA binding does what?
Stimulates replication and decreases replication
What stimulates replication?
Low concentration of plasmid
What decreases replication?
High concentration of plasmid; Plasmids coupled
What are the qualities of a good plasmid for cloning vectors?
- ori
- a selectable marker
- a multiple cloning site (MCS) inside a scorable marker
- small
- high copy number
The concept of ____________ is important because it is a quick easy, 1-step process of determining whether a transformed bacterial colony has plasmid + insert or not.
Alpha-complementation
The key to alpha-complementation is?
The fact that the lac-Z gene product is a tetramer, and each monomer is made of two parts. lacZ-alpha, and lacZ-omega.
What is another method of alpha complementation without using a alpha- complement?
Completely delete the lac operon from the chromosome, but introduce lac-Z-omega fragment on an F’ fertility factor
When a bacteria lose the plasmid during cell division, they are said to be what?
Cured of the plasmid
What are the 3 mechanisms to prevent curing of plasmids
1: Addiction systems (plasmid produce toxic protein that kills the cells that lose the plasmid)
2. Site-specific recombinases
3. Partitioning systems
Multimer resolving enzymes are what?
Site-specific recombinases
What normally acts at the dif site to resolve replicating chromosomes?
XerCD
XerCD can also act at the ________ site in ColEl plasmids to resolve plasmid dimers.
Xer
P1 plasmid system of E. coli is a prototype comprising a partition site and two proteins, ParA and ParB1. To form the partition complex necessary for segregation, what must happen?
P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of 74bp.
A crucial step in partition is the initial formation of the so-called “__________” between P1 ParB and the partition site.
partition complex
Partition complexes are bound by what? (which delivers each plasmid to its correct cellular location)
ATPase ParA
ParB of E. coli consists of a proteolytically sensitive __________ (residues 1-141), which binds _______ and forms higher order oligomers and a carboxy-terminal dimerization region (residues 142-333), which contains all of the determinants required for DNA binding.
N-terminal region; ParA
The ________site is the minimal partition site required for segregation.
parS-small
Partition efficiency is increased when full-length ______ is used and the site is bent by the host auxiliary factor IHF.
parS
P1 plasmid system of E. coli is a prototype comprising a partition site and two proteins, ParA and ParB1. To form the partition complex necessary for segregation?
P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of 74 bp.
