Transposons and Retrotransposons Flashcards
Explain what is meant by Transposable Elements or TEs.
Transposable elements (TEs) are DNA sequences that can change their position within a genome
Contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material
Active elements but many dead elements which cannot move
What are the genetic consequences of TEs?
Insertion of TEs may activate or disrupt a gene upon movement.
Can promote re-arrangements by providing dispersed regions of homology
Describe how TE’s effect Maize.
Bz = bronze locus in wild type maize
Point mutation leads to bz - paler colour
TE insertion = bz-m leads to paler colour with bronze spots (wild type colour)
Transposons can cause unstable alleles - high rates of reversion
Describe how TEs are involved in hybrid dysgenesis in Drosophila.
If egg has no defense against TEs and sperm has TEs, offspring will be highly mutated
Descendants in a few generations will be immune to those specific TEs
Some strains have TEs in egg so doesn’t suffer any consequences by being fertilised by sperm of another strain
What is the basic minimal insertion sequence structure for transposons aka simple transposons?
Inverted repeats (genetically required, in cis) which flank TNP (transposase) gene (genetically required, trans-acting)
Short direct repeats (typically few bp) but are generated at time of insertion and aren’t part of transposon
Any piece of DNA can move around as long as it has inverted repeats at either end and a transposase encoded somewhere in the genome
What are DDE transposons?
Best characterised
Gene is cut out and pasted into target
1. Transposase binds to ends of inverted repeats and cuts it out of host molecule by two tranposase molecules coming together
2. Sticks in somewhere else (excisive transposon)
3. Staggered DSB in target DNA (nicks aren’t opposite each other)
4. Transposase sticks transposon within region of the nicks, resulting in strand of ssDNA
5. DNA Polymerase replicates ssDNA
Describe the ways in which transposons are important in biotechnology.
NGS library preparation:
Tagmentation of sequence of interest
Add primers and amplify for cluster generation
Transposon tagging in vivo for gene isolation:
Put a transposon into a genome and get it to move by turning on transposase
Select for mutant
Sequence mutant and find where transposon has landed
Have gene without having to know anything about it beforehand
Describe the ways in which excisive transposons spread.
- Jump ahead of replication fork
Most likely; start with 2 elements and end up with 3 - Jump in G2 and use sister which still has transposon for repair (link to BFB cycles)
What is meant by the term degenerate transposons?
Caused by interruptions in the DSB repair process
Often have the terminal repeats (wheels) but lack the transposase (engine)
Sometimes both wheels and engine aren’t working
True of both replicative and excisive
e.g. AcDs element - Ac (activator) can mobilise Ds (dissociator)
What are Replicative Mechanisms of transposons?
Original cut of transposon is only a nick and only one strand at each end is initially ligated
3’ OH groups look like primers for DNA replication and function as such
Element is replicated and circular co-integrate structure is produced as an intermediate
Co-integrate is resolved by resolvase into 2 separate molecules
Copy and paste
What are the two types of retrotransposons?
- Long Terminal Repeats (LTR): like retroviruses but cannot move between cells
- Reverse Transcriptase/endonuclease (RT/en): Used to be called non-LTR; can be autonomous and non-autonomous
What is the life cycle of a retrovirus?
LTR arranged U3 - R - U5 either side of coding region
mRNA translated into polyproteins for gag (Matrix, Capsid, Nuclear Binding Protein), and pol (Protease, RT and Integrase) - proteolytically digested into functional proteins for viral nucleocapsid, RTase and integrase
Spliced to produce message for env (surface proteins for viral nucleocapsid, RTase and integrase)
Virus fuses with membrane
Nucleocapsid is released
dsDNA transported through nuclear pore into nuclease using integrase
Provirus integration by integrase making a nick in DNA (establishes lifelong infection - HIV)
Transcription and translation
Exocytosis: Cluster of envelope proteins shuttled from RER to outside of infected cell
Polyprotein chains bind to inside PM
Immature virion buds off with envelope proteins on outside
Protease breaks polyprotein chains allowing them to coalesce and form mature structures
Mature virion can infect other cells
What are LTR Retrotransposons?
Can be thought of as transposons that have learnt how to convert their mRNA into DNA - use DDE enzyme as their integrase
Similar to retroviruses but lack envelope proteins
e.g. Yeast Ty elements contain gag and pol but not env genes
Not active currently in human genome but evidence for recent activity
Empty LTRs arise by homologous recombination and some are unique to humans
What are the activities of reverse transcriptase?
- RNA template mediated DNA synthesis; reverse transcription-RNA primed
- DNA template mediate synthesis - RNA primed
- RNAase H degrades RNA in RNA/DNA hybrids - only in ds, endonuclease, liberates short oligonucleotides
Describe how ssRNA is reverse transcribed into dsDNA.
Genome in viral particle with primer bound at PBS
DNA synthesis continues as far as 5’ end
Now have an RNA/DNA hybrid (because template is RNA)
RNA Hase activity degrades RNA
5’ R is complementary to 3’ R so anneals to that sequence
Primer can prime more DNA synthesis extending to PBS
As RTase is working, the RNAase H is degrading the template behind it
Polypyrimidine tract is refractory to activity of RNAase H (not degraded)
These RNA molecules left behind prime synthesis of second strand
DNA ends up copying sequence of tRNA which then gets degraded
This ssDNA is complementary to PBS so anneals to first PBS
Circular structure
Continue synthesis and strand displacement to give dsDNA where LTRs are replicated at either end
