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molecular genetics > The Extrachromosomal > Flashcards

The Extrachromosomal Flashcards

1
Q

how does adenovirus replication occur?

A
  • the virus is double stranded and linear
  • both ends are annealed and replication begins with the bottom strand
  • the top strand is displaced and because it is an inverted repeat, it is able to mimic a double strand by turning into a frying pan structure
  • the terminal protein believes it is double stranded
  • the terminal protein and the primer dCTP binds to the 5’ end of the adenovirus DNA
  • NF1 (hijacked host protein) is able to bind to the primer and signal where an initiation complex can form and synthesis can begin
  • Oct1 stabilizes NF1, DNA Pol, and helicase
  • DNA Pol binds and host helicase unwinds
  • the terminal protein is still left behind
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2
Q

what is the role of the terminal protein?

A
  • first nucleotide is fused to cytosine which acts as a primer
  • DNA polymerase is now able to bind covalently and is anchored from the terminal protein
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3
Q

NF1

A
  • essential for initiation
  • transcription factor is hijacked by TP to stabilize it so the virus DNA polymerase can transcribe
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4
Q

how does the displaced strand mimic the double-stranded linear adenovirus?

A
  • The first nucelotides have the same sequence, so they reattach at the 5’ end and the 3’ end to mimic the beginning of a double stranded DNA
  • The TP comes and replaces a G to a C to begin the process of transcription and the rest is explained above
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5
Q

bacterial conjugation

A
  • tip of the f pilus makes contact with the recipient cell (if positive, no connection is formed this is controlled via TraS and TraT)
  • the pilus retracts bringing recipient closer for transfer
  • transfer begins from the oriT which is nicked by TraY/TraI complex, traI nicks
  • DNA transferred through the T4SS channel - the replicated DNA is is guided to the channel via TraD - Tra N and Tra G may also participate in this process
  • a plasmid genome or hostc chromosome is transferred from one bacterium to another in a mating process mediated by F plasmid
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6
Q

what is the pilus composed of?

A
  • composed of pilin subunits which form a hollow cylinder of 8 nm with 2 nm inner diameter. (ssDNA can move through pores as small as 1 nm)
  • used to think it was ds so the DNA must have integrated outside of the pilus
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7
Q

T4 Secretory System

A
  • T4 was first found in T4 phages and thought to only be used by them but now we know that it is used to transfer many things, in conjugation it is used to transfer DNA
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8
Q

TraD

A
  • active transport
  • binds DNA
  • ATP/GTP binding sites, inner membrane protein
  • necessary for DNA transfer
  • directs 5’ end to the T4SS
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9
Q

TraI

A
  • has nuclease and helicase (ATP) activity
  • function enhanced by traY and IHF (integration host factor)
  • is a transfer (pilot protein)
  • covalently attaches to the 5’ end of the DNA after cutting one strand
  • Relaxase because it relieves the super coiled tension by nicking the strand at the origin to be released
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10
Q

TraY

A

binds near oriT and recruits Tra I to the 5’ end

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11
Q

F plasmid

A
  • also called the F factor
  • fertility plasmid
  • transferred by conjugation between bacteria
  • A free F factor is a replicon that is maintained at the level of one plasmid per bacterial chromosome
  • codes for pili that form on the surface of the bacterium
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12
Q

what are the three methods of replication in F plasmids?

A
  • oriV as free plasmid (one copy/bacterial chromosome)
  • Uses E. coli chromosomal origin when integrated (oriV suppressed)
  • oriT during conjugation
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13
Q

what is the mechanism behind the tra region?

A
  • tra J binds to the upstream region that then acts as a positive promoter for Tra M and the polycistronic promoter and regulated by fin P
  • Tra M then regulates whether a mating pair has formed or has formed correctly
  • Tra Y and I form a heterodimer - TraY binds to the oriT but cannot cut until TraI is produced
  • TraI waits for TraY to bind to the origin so it can cut
  • tray/trai multimeric complex migrates around the circle and unwinds DNA from 5’ end at 1,200 bp/sec
  • only one strand is produced
  • once complete, TraD brings the product through the channel
  • TraS and TraT control immunity so the cell does not infect a cell that already has the F plasmid
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14
Q

tra region

A
  • how all plasmids transfer themselves
  • transfer region
  • has a regulatory region and a series of genes in RNA - polycistronic RNA
  • there are about 40 genes in the tra locus
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15
Q

IS elements

A
  • simple transposons encode transposase; used to insert into host chromosome (integration)
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16
Q

transposons

A
  • very simple genetic elements comprised mainly of the enzyme responsible for their jumping
  • Sometimes can code for other proteins
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17
Q

what are the types of transposition can transposons go through?

A
  • replicative transposition
  • nonreplicative transposition
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18
Q

replicative transposition

A

make a copy of themselves in the process of jumping - moving to another DNA

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19
Q

nonreplicative transposition

A
  • leaves a double stranded break and jumps to another position that already has an IS element the same as itself
  • Can sometimes end up integrating the f plasmid as they jump - take the entire f plasmid with them
  • caused by homologous recombination
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20
Q

TraM

A
  • sense a mating pair has formed
  • sees mating type and status
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21
Q

TraT

A

outer membrane protein that block mating pair formation

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22
Q

TraS

A

blocks DNA transfer

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23
Q

TraJ

A
  • regulator protein it binds to the promoters of tra M and the ploycistronic promoter
  • acts as a positive acting transcription factor - the promoters that it regulates does not act or work unless it is bound to the upstream regions of the promoter
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24
Q

TraD

A
  • binds to the end and brings it to the channel through the pilli
  • is apart of the channel subunit
  • coupling protein
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25
Q

finP

A
  • Negative regulator transcript
  • Makes an anti-sense RNA - opposite code that codes for the protein
  • Shuts down/regulates the amount of tra J
  • Type of feedback control
  • Does NOT encode a protein
26
Q

surface exclusion

A
  • Immunity
  • If f+ there will be no conjugation
  • Coded by traS and T
27
Q

episome

A
  • a genetic unit (phage, plasmid) that is integrated or can be integrated into the host genome
  • example is F plasmid
28
Q

oriV

A
  • Vegetative replication
  • When the f plasmid is floating in the cell and not wanting to replicate
  • Free plasmid (one copy/bacterial chromosome)
  • Origin where DNA replication initiates when the f plasmid is in its free form
  • Used to initiate plasmid replication
29
Q

oriT

A
  • Site where transfer begins
  • A nick is put in to start rolling circle replication
  • Used to initiate replication for transfer (in conjugation-rolling circle model)
  • The minimum you need to duplicate genes
30
Q

pilin

A
  • identical subunits that polymerize and extend and make contact. Then depolymerize to pull the two cells together
  • inside the pilus
31
Q

mob/bom site

A
  • When origins or sites where y and i make the nic are called different things
  • bom = basis of mobilization
  • mob = mobility site
32
Q

incompatibility group

A

A group of plasmids that cannot mutually exist within a cell, overtime one will out compete the others; usually the smaller one because it has a better efficiency of replication
- incompatible because they share a common oriV
- usually the smallest one will win because it can replicate faster

33
Q

what are incompability groups for F plasmids and RP4 plasmids?

A
  • F = IncF
  • RP4: IncP
34
Q

promiscuous plasmid

A
  • In conjugation there are groups with some tra regions that can act universally and work with a wide range of bacteria
  • Can recognize a wide range of OriT
35
Q

mob plasmid

A

small plasmid you want to engineer and transfer to another organism

36
Q

helper plasmid

A
  • to enable a small cloning plasmid to go into a recipient cell, a helper plasmid is used
  • delete the oriT on the helper plasmid and engineer an oriT onto the cloning plasmid
  • may or may not be transmissible (depends if it has an oriT)
37
Q

what is the minimum needed to be transferred to another cell?

A

an oriT

38
Q

HFR cell

A
  • High frequency recombination strains
  • When F+ is not integrated into the cell
  • Had F plasmid but was integrated and not a free plasmid
  • F plasmid was trying to integrate but could not because it was transferring the chromosomal DNA first before its plasmid
39
Q

how long does it take to transfer the entire E. coli genome before the F plasmid can finish replicating itself?

A

100 minutes

40
Q

F prime plasmid

A
  • Plasmids that leave the genome carrying chromosomal DNA are known as prime factors
  • They leave the chromosome by homologous recombination, resulting in the deletion in the chromosome
  • Instead of being inserted into host genome it will leave the host and will take part of the host chromosome with it
41
Q

what protein is similar to TraY and TraI

A

protein A

42
Q

what are the two mechanisms of DNA integration?

A
  • homologous recombination
  • transposition
43
Q

what are plasmids?

A
  • self-replicating circular genomes that maintain a relatively constant copy number
44
Q

low copy

A
  • from 1-10 copies per cell; have segregation mechanism
45
Q

high copy

A

more than 10 copies, sometimes ip to 100 or more
- are distributed between the daughter cells randomly

46
Q

episome

A

a plasmid that can integrate into the host chromosome
- fertility plasmid

47
Q

what are phages?

A

self-replicatig entities that produce infectious particles
- may be circular or linear and may be DNA or RNA

48
Q

lysogenic

A

phages that can integrate into the host genome

49
Q

immunity

A

the ability of a phage or plasmid to exclude reinfection by a similar entitiy

50
Q

how can phages and plasmids sometimes mediate the exchange of host genes?

A
  • when phages and plasmids exit the host genome, the host DNA can be taken in the process
  • phages transfer through infectious particles while plasmids transfer through conjugation
51
Q

what are some issues involved with linear replication?

A
  • DNA polymerase surrounds the origin
  • terminal proteins
  • strand displacement with unusual structure
  • repetitive sequences
  • rolling circle replication
52
Q

in adenovirus replication, which proteins are the virus and which proteins are the host?

A
  • virus: terminal protein, DNA polymerase, and DNA binding protein (helicase)
  • host: NF1 and Oct 1
53
Q

rolling circle replication

A
  • single stranded circle packaged into an infectious particle
  • breaks out of packaging once in the recipient
  • needs to be turned into a double stranded circle in order to make copies because it needed to be in replicative form - this is called lagging strand synthesis
  • once a double stranded circle, protein A can bind to the origin and cuts the outside strand and binds to the 5” end (like a terminal protein)
  • the 3’ end in bounded to DNA polymerase for synthesis
  • once back to the origin, protein A cuts the new segment and ligases it
  • then put into virion packaging and can infect new cells
  • this second method is called leading strand synthesis
54
Q

what are the functions of protein A?

A
  • origin recognition
  • endonuclease
  • ligase
  • protein A is a cis-acting relaxase that generates a single circle from the tail produced by rolling circle replication
55
Q

what is rolling circle used by?

A

plasmids to transfer themselves and host chromosome to other bacteria during conjugation

56
Q

a free F factor

A

a replicon that is maintained at the level of one plasmid per bacterial chromosome

57
Q

F factor (plasmid)

A
  • can integrate into the bacterial chromosome and its own replication system is suppressed
  • codes for pili that form on the surface of the bacterium
  • large circular plasmid (100 kb)
  • 32 kb is organized into the transfer region
58
Q

tra A

A

codes for the protein pilin that is used to make the connection via the pilus in bacterial conjugation

59
Q

self-transmissible plasmid

A

contains a tra region and an oriT

60
Q

mobilization plasmid

A

only contains an oriT
- mob or bom site

61
Q

what does an hfr cell contain?

A

an integrated F plasmid

62
Q

what does an F+ cell contain?

A

episomal F plasmid

molecular genetics (28 decks)

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