The Extrachromosomal Flashcards
how does adenovirus replication occur?
- 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
what is the role of the terminal protein?
- 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
NF1
- essential for initiation
- transcription factor is hijacked by TP to stabilize it so the virus DNA polymerase can transcribe
how does the displaced strand mimic the double-stranded linear adenovirus?
- 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
bacterial conjugation
- 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
what is the pilus composed of?
- 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
T4 Secretory System
- 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
TraD
- active transport
- binds DNA
- ATP/GTP binding sites, inner membrane protein
- necessary for DNA transfer
- directs 5’ end to the T4SS
TraI
- 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
TraY
binds near oriT and recruits Tra I to the 5’ end
F plasmid
- 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
what are the three methods of replication in F plasmids?
- oriV as free plasmid (one copy/bacterial chromosome)
- Uses E. coli chromosomal origin when integrated (oriV suppressed)
- oriT during conjugation
what is the mechanism behind the tra region?
- 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
tra region
- 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
IS elements
- simple transposons encode transposase; used to insert into host chromosome (integration)
transposons
- very simple genetic elements comprised mainly of the enzyme responsible for their jumping
- Sometimes can code for other proteins
what are the types of transposition can transposons go through?
- replicative transposition
- nonreplicative transposition
replicative transposition
make a copy of themselves in the process of jumping - moving to another DNA
nonreplicative transposition
- 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
TraM
- sense a mating pair has formed
- sees mating type and status
TraT
outer membrane protein that block mating pair formation
TraS
blocks DNA transfer
TraJ
- 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
TraD
- binds to the end and brings it to the channel through the pilli
- is apart of the channel subunit
- coupling protein