Lecture 5 - Retroelements Flashcards
Define retroelements
mobile elements that replicate through an RNA intermediate, use reverse transcriptase to sythesise DNA from RNA
What are the two major types of retro elements?
LTR transposons
non-LTR transposons
What are the properties of LTR transposons? (e.g. Ty in S.cerevisae)
contain LTR
use reverse transcriptase and/or integrase
What are the properties of non-LTR retrotransposons? (e.g. L1 in human)
don’t contain repeats
use reverse transcriptase/endonuclease
What are the properties of SINES? (SINES in mammals)
no repeats
no enzyme activites
no introns
What are similarites between LTR retroelements and retroviruses?
both contain -LTR -integrase -usually gag gene all encode reverse transcriptase non have introns as passage through mRNA would splice them out
What is the mechanism of replication of the retroviral genome?
1) reverse transcriptase makes cDNA copy of the RNA genome, bound by LTR
2) cDNA copy transported into nucleus and integrated by integrase randomly
3) transcription and RNA virus packaged into viral particles
How is the RNA form of the virus different from the integrated provirus or cDNA copy?
- in cDNA LTRs comprise U3_R_U5
- RNA copy 5’ end is R_U5 and 3’ end is U3_R
How does the 5’ end of the RNA aquire a copy of U3 and the 3’ end aquire a copy of U5?
cDNA copying
What is the mechanism of cDNA copying?
- Host tRNA used to prime cDNA synthesis using host RTase at the primer binding site downstream of U5
- first strand cDNA synthesis copies viral RNA to the end generating a RNA c/DNA hybrid molecules composing PBS, U5 and R regions.
- RNAseH then digest the RNA of the hybrid molecule
- newly syntheised cDNA peels off PBS and rebinds via the complimentarity at the R region of the 3’ end of viral RNA (first jump)
- first strand cDNA synthesis continues to end of the molecule completeing the right LTR in cDNA strand (U3_R_U5)
- RNAseH digests most of RNA leaving only fragments hybridised to first strand cDNA copy
- hybridised RNA fragments act as primers as generate second cDNA strand of the right LTR and PBD
- tRNA acted as primer for first strand cDNA synthesis removed.
- second cDNA strand is peeled off and rebinds to PBS in the left end of the first stand cDNA (second jump)
- DNA polymerases complete DNA synthesis of first cDNA copy to form left LTR and of the second cDNA to copy main body of viral genome and right LTR
What is the mechanism of viral cDNA integration into host chromosome (in relation to HIV)?
- Integrase binds to the LTRs and nicks the DNA to create a 3’OH group
- Integrase uses 3’OH groups to attack DNA target site randomly in chromosome
- attack by 3’OH neucleophile cleaves target and joins 3’OH in HIV cDNA to 5’ end of target site
- gaps created by staggered break repaired by host enzmyes
What is the structure of the Ty element?
LTR - tyA (gag) - tyB (pol) - LTR
What does TyA gene encode for, and what does frameshift at end of TyA lead to?
TyA = capsid protein
Framshift leads to TyA-B protein = reverse transcriptase, protease, integrase
What experiment was done to show that transposition of Ty element requires an RNA stage and resembles retrovirus replication?
Ty element was engineered:
-marking LTR by mutation
-inducible promotor (GAL) added upstream of Ty element
-intron within one of the ORFs
-introduced into yeast on a plasmid
Two Experiments
1. Engineered recombinant Ty in plasmid vectors
2. Trasnform yeast cells with plasmid vectors and grow in either galactose or nongalactose containing media
Results
galactose -
1.TymRNA synthesis increased
2. Transposition of Ty elements increased
non-galactose -
1. Ty mRNAs lacked intron
2. transported Ty elements lack intron
What did the transposition of Ty element in yeast experiment show?
- induction of Ty element transcription resulted in multiple copes of the new Ty in chromosome
- all new copies lose intron - spliced out before mRNA is copied into cDNA
- transposition dependent on transcription
- new copies have identical LTR
