Transposition 1 Flashcards
what can transposition events cause?
- may cause deletions or inversions or lead to movement of host sequences to new locations
- can mix up the genome or move host sequences to new locations
transposons
- genetic elements in the genome that are mobile
- A major cause of variation in almost all genomes is caused by transposable elements and their presence can have a major impact on the overall size of the genome
- Transposons are DNA elements that “hop” or transpose to different places within the genome
what part of the genome is comprised of transposons in maize and corn?
- roughly half
- Usually nonfunctional, transposons is no longer active
- Effort is suppressing transposons
what are the two major classes of transposons?
retroelements and DNA-type elements
what are retroelements?
- make DNA copies of their RNA transcripts
- Encode a unique enzyme required for their transposition (reverse transcriptase)
- primarily in eukaryotes
what are the types of retroelements?
LTR retrotransposons, Retroviruses, and nonLTR-retroposons
what are DNA-type elements?
- Directly propagate as DNA by jumping to new sites within the host genome.
- Encode transposase enzyme
- Sometimes will carry a second gene to dissolve a cointegrate between circular genomes that are a result of replicative transposition - resolvase
- If they only make copies of themselves it is only transposase
- common in both eukaryotes and prokaryotes
- do not affect anything other than DNA
- nonhomologous recombination
- only transport their own sequence - no other DNA
LTR retrotransposons
- have long terminal repeats
- no infectious particle
retroviruses
- closely related to LTR but produce a coat protein to form an infectious particle
- also has LTRs
non-LTR retrotransposons
- have a different mode of replication than LTR retrotransposons
what are the ways transposition can occur?
- generate a new copy at a random site
- or the transposon may transpose directly and leave a hole in the donor DNA
what affect can transposition have?
- Transpositions to a new site can have a major impact on genome structure
- Unequal crossover events between related transposons can cause rearrangements in the order of genes
transposition
the movement of a transposon
transposase
- enzyme that cits and ligates DNA
where were transposons first identified?
in bacterial operons-spontaneous silencing
what is the simplest transposon?
IS elements
what are the two types of transposition?
replicative and nonreplicative
what is the name of sequences that flank the transposase gene in a simple transposon?
ITR
what is the rate of transposition?
10^-4
- rate of a particular gene picking up a transposon is about equal to rate of a gene undergoing spontaneous mutation
what is the rate of spontaneous mutation?
- 10^-5 to 10^-7 per generation
what is the rate of reversion?
10^-6 to 10^-10 per generation
- reversion is 10^3 times less frequent than insertion
what are ITRs?
- inverted terminal repeats
- define the ends of transposon, closelt related rather than identical
- the recognition of these ends is critical in transposition and point mutations abort it
- transposase is encoded by sequences within the transposon and between the ITRs
what is the process of insertion elements?
- staggered nicks are made at the target site
- transposon joined to single-stranded ends
- gaps at target site are filled in and sealed through direct repeats
staggered ends
- As a consequence of jumping, transposase lands on the target and cuts its own 3’ end and ligase to the target site and cutting the target and separating it.
- Does not make a staggered end cut as an offset between the two ends. The amount of bases is the geometry of the transposase
- varies between 4-13
direct repeats
DNA repair on both sides to restore DNA after the staggered ends have been created; repair reaction
length constant
the amount of bases of direct repeat generated in the target
- how far the cut is staggered
what are composite transposons?
- contain chromosomal DNA flanked by two IS elements of the same type
- the IS elements onlt carry enzymes needed for transposition
- central region may carry drug resistance
- flanking IS elements can be in either orientation
What constitutes arms of a composite transposon?
Composite transposons contain chromosomal DNA flanked by two IS elements of the same type
are both arms of a composite transposon functional?
Composition transposons usually only one arm has a functional transposase
Only two of the IRs need to be cut for transposition
direct repeat of IS elements
- Direct repeats of the IS element recombine to excise material
- Two identical IS elements oriented in direct repeats (or inverted) when recombination occurs, they gene needs to be twisted around for recombination to occur
- This cuts out one of the transposons and everything in between
- Directly excise all the materials in between
inverted repeat recombination
- inverts material
- When the IS elements are lined up, the sequences are flipped, and homologous recombination occurs, you invert the sequences between the two IS elements
- Inverted repeats result in inversion of the transposon (or any material between direct repeats)
what can the loss of a transposon result in?
the restoration of gene function at the site of insertion
precise excision
requires removal of one copy of the transposon and one of the direct repeats at the target site
- rare event that probably involves recombination between the two copies of the target site direct repeats
Replicative transposition
- proceeds through a cointegrate structure
- Fusion of two original molecules; requires host replication machinery
- proceeds through a strand crossover complex
- requires site-specific recombination in order for the transposon to be on both copies of DNA
- Replicative transposition involves attack of the 3’ end of the donor molecule to the target. It cuts and joins to the target at both ends of the transposon (donor)
- A replication fork forms at one of the ends and extends the 3’ end of the target for leading and lagging strand synthesis
- the resolution requires the transposon-encoded enzyme resolvase acting at the res site
what are the two models for nonreplicative transposition?
cut and paste model (cleavage and ligation) and break and reunion model
nonreplicative transposition - cut and paste model
- Transposon is excised free of the donor and the 3’ ends attack the recipient DNA
- Ligate it to the target cut site - 3’ end of transposon is ligated to 5’ end of the target
- Repair of the gaps formed at both ends in order to connect donor and target DNA for transfer of transposons
- Since already cut, there is a repair operation to generate the direct repeat
- Cut both strands as with replicative only one strand is cut until the resolvase step
- transposon is then transferred to target DNA
break and reunion model
- Tn5 shows a different type of double strand cutting mechanism involving hairpin formation
- Tn5 is cleaved from flanking DNA
- Tn5 and Tn10 transposases act as dimers
- each subunit has an active site that creates ds cut at the end of the transposon and then creates a staggered cut at the target site
- the geometry of the dimer determines the length of the offset in cuts
what are the two outcomes of composite transposition?
- Two IS10 modules can mobilize any region of DNA that lies between them
- Transposon integrates into circular DNA
- Transposon can move again back into host chromosome and there are two possible outcomes
- If the inside cut direct repeat, the plasmid genes will move
- The unit that moves is what contains the intact terminal repeats
- New transposon configuration created by mobilization of - IS10 module in alternative orientation
- If cut on the outside of the direct repeat, the IS elements and gene will move
Which ends of the donor attack the target DNA and covalently join the target?
3’ end of the donor attack the 5’ end of the target DNA
In the Strand Crossover Complex, which ends serve as primer for DNA synthesis?
3’ end of the target serves as a primer for DNA synthesis
