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Prokaryotic Molecular Genetics > Plasmids > Flashcards

Plasmids Flashcards

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1
Q

What are Plasmids?

A

small, circular, extra-chromosomal DNA which replicates independently of host chromosomal DNA

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2
Q

Are all plasmids circular?

A

No, they are not.

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3
Q

R-factors in plasmids give bacteria what?

A

Antibiotic resistance

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4
Q

How many proteins can plasmids encode for?

A

Depending on their size, they can encode either a few or hundreds of different proteins

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5
Q

Do plasmids give large amounts of advantages to bacteria?

A

No, they give bacteria selective advantage only in certain conditions

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6
Q

When purifying plasmids, after hours of ultracentrifugation, where in the tube would you find the plasma DNA?

A

Due to a formation of a gradient, the plasmid would be the lower band showing

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7
Q

The EtBr bound to DNA makes it ___________ in salt solution made with heavy atoms such as CsCL.

A

less dense

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8
Q

What are the 3 properties of plasmids?

A
  1. Replicon (DNA molecules that can replicate autonomously in the cells
  2. Plasmids has one origin of replication (oriV)
  3. The ori region determines the mech. of replication
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9
Q

What are the two types of plasmid replication?

A
  1. Theta replication

2. Rolling circle

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10
Q

What is Theta replication?

A

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)

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11
Q

What is unidirectional replication?

A

Replication that terminates when the replication fork gets back to the origin

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12
Q

What is bidirectional replication?

A

Replication that terminates when the replication fork meets somewhere on the DNA molecule opposite the origin

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13
Q

Rolling circle plasmid replication for this mechanism are named ________.

A

RC plasmid

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14
Q

There are two stages to rolling circle plasmid replication. What are they?

A
  1. Replicated and form double and single strand circular plasmid
  2. The complementary strand is synthesized on the single-stranded DNA to make another double-stranded DNA
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15
Q

What kind of protein is encoded by the plasmid?

A

Rep protein

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16
Q

What are the functions of the Ori regions?

A
  1. Host range

2. Regulation of copy number

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17
Q

When dealing with the Host range of functions, what are the ranges seen as functions of the Ori regions?

A
  1. Narrow host range
  2. Broad host range
  3. Incompatibility
  4. Plasmid replication control mechanism
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18
Q

What regulation of copy number functions of the Ori region is?

A

Relaxed plasmid, molecules which inhibit a plasmid replication when the number reach certain level (mechanism present in high copy plasmids

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19
Q

Plasmid incompatibility is when plasmids ____________, and one or the other will be lost at a higher than normal rate when the cells divide

A

can not coexist

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20
Q

What is Incompatibility group (Inc group)?

A

It is the plasmid members of the same Inc group that are not able to coexist

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21
Q

Why can’t plasmids members of the same Inc group are not able to coexist?

A

Because they share the same replication control mechanism and/or the same partitioning (par) functions

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22
Q

When dealing with plasmid replication control mechanism, what type of plasmid is regulated by a ctrRNA and Protein?

A

Cole1-derived plasmid

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23
Q

R1 and ColB1-P9 plasmid does what in bacteria?

A

Regulation of Rep protein by antisense RNA

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24
Q

How are Iteron plasmids regulated?

A

Regulation by Coupling

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25
Q

What type of plasmid are ColE1-derived plasmid?

A

Endogenous plasmid

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26
Q

ColE1-derived plasmid makes _______ protein

A

Immunity

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27
Q

Many plasmid cloning vector are derived from what type of plasmid?

A

ColE1-derived plasmid

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28
Q

ColE1-derived plasmid replication is regulated by?

A

RNA I (small RNA encoded by plasmid)

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29
Q

In ColE1-derived plasmid, RNA I interfere with the processing of RNA II by?

A

forming a double strand RNA.

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30
Q

RNA I and RNA II are ___________.

A

complementary

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31
Q

What forms the primer for plasmid replication in regards to ColE1-derived plasmid?

A

RNA II

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32
Q

Do ColE1-derived plasmid require encoded proteins to initiate DNA replication at their oriV?

A

No, they do not require encoded protein.

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33
Q

Imm stands for?

A

Immunity protein

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34
Q

Kil stands for?

A

lyses protein

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35
Q

Inc stands for?

A

RNAI (ctRNA)

36
Q

rnall stands for?

A

replication primer

37
Q

oriV stands for?

A

origin

38
Q

Rop does what?

A

regulates copy number

39
Q

Bom stands for?

A

origin of transfer

40
Q

Mob stands for?

A

transfer protein

41
Q

Cer does what?

A

maintains monomers

42
Q

What does Exc do?

A

excludes incoming plasmids

43
Q

What does Cea mean?

A

Colicin toxin

44
Q

What are the two mechanisms of regulation?

A
  1. Stop replication

2. Initiate replication

45
Q

How does stop replication work? (Mech. of Regulation)

A

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

46
Q

How does initiate replication work? ( Mech. of Regulation)

A

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

47
Q

More plasmid –> __________ –> stops replication

A

more Rop + RNA I

48
Q

Less plasmid –> ____________ –> allows replication

A

les Rop + RNA I

49
Q

Rop + RNA I —> ?

A

no plasmid replication

50
Q

RNA II –>?

A

plasmid replication

51
Q

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

A

True

52
Q

What type of plasmid-encoded protein do most plasmids require?

A

Rep

53
Q

What does “Rep” do?

A

It separates the strand of DNA at oriV region

54
Q

Is “Rep” specific?

A

It is very specific, only binds to oriV from the same plasmid –> recognize specific sequences

55
Q

T or F: The synthesis of Rep fully controls the copy number of the plasmid

A

False: it only partially controls the number of plasmid copies

56
Q

What is done to inhibit the synthesis of Rep protein?

A

An antisense RNA (called CopA)

57
Q

How does antisense RNA work?

A

Antisense RNA forms a hybrid

58
Q

Hybrid prevents translation of what?

A

Rep A

59
Q

What does RNase III target?

A

The Hybrid

60
Q

T or F: RNA III is a chromosomal enzyme

A

True

61
Q

When does replication occur?

A

Replication occurs after plasmid enters the cell

62
Q

What is “iteron sequence”?

A

It is an oriV region that contains several repeats of a certain set of DNA bases

63
Q

The interon sequence has ___ to ___ bp long and exist from ___ to ___ copies in this region.

A

17 to 22; 3 to 7

64
Q

What does repA gene encode for?

A

RepA protein

65
Q

What makes RepA protein so important?

A

it is the only plasmid-encode protein required for replication

66
Q

The host chromosome encodes the other proteins that bind to this region to allow initiation of the replication. What are they?

A

DnaA, DnaB, DnaC, and DnaG

67
Q

RNA binding does what?

A

Stimulates replication and decreases replication

68
Q

What stimulates replication?

A

Low concentration of plasmid

69
Q

What decreases replication?

A

High concentration of plasmid; Plasmids coupled

70
Q

What are the qualities of a good plasmid for cloning vectors?

A
  1. ori
  2. a selectable marker
  3. a multiple cloning site (MCS) inside a scorable marker
  4. small
  5. high copy number
71
Q

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.

A

Alpha-complementation

72
Q

The key to alpha-complementation is?

A

The fact that the lac-Z gene product is a tetramer, and each monomer is made of two parts. lacZ-alpha, and lacZ-omega.

73
Q

What is another method of alpha complementation without using a alpha- complement?

A

Completely delete the lac operon from the chromosome, but introduce lac-Z-omega fragment on an F’ fertility factor

74
Q

When a bacteria lose the plasmid during cell division, they are said to be what?

A

Cured of the plasmid

75
Q

What are the 3 mechanisms to prevent curing of plasmids

A

1: Addiction systems (plasmid produce toxic protein that kills the cells that lose the plasmid)
2. Site-specific recombinases
3. Partitioning systems

76
Q

Multimer resolving enzymes are what?

A

Site-specific recombinases

77
Q

What normally acts at the dif site to resolve replicating chromosomes?

A

XerCD

78
Q

XerCD can also act at the ________ site in ColEl plasmids to resolve plasmid dimers.

A

Xer

79
Q

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?

A

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.

80
Q

A crucial step in partition is the initial formation of the so-called “__________” between P1 ParB and the partition site.

A

partition complex

81
Q

Partition complexes are bound by what? (which delivers each plasmid to its correct cellular location)

A

ATPase ParA

82
Q

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.

A

N-terminal region; ParA

83
Q

The ________site is the minimal partition site required for segregation.

A

parS-small

84
Q

Partition efficiency is increased when full-length ______ is used and the site is bent by the host auxiliary factor IHF.

A

parS

85
Q

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?

A

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.

Prokaryotic Molecular Genetics (13 decks)

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