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
What are LINES?
Non-LTR retroposons
Long interspersed nuclear elements
What is the structure of LINES?
contain two ORF
ORF1 = contains DNA binding protein
ORF2 = encodes reverse transcriptase and an endonuclease
Both are required for transposition
What is the proposed mechanism for LINE copying? (coupled endonucleolytic/replicative strategy)
- ORF2 protein binds and L1 endonuclease nicks target DNA creating a 3’OH which captures L1 RNA at base pairing
- L1 reverse transcriptase used 3’OH on nick site as a primer a synthesises L1 cDNA
- Reverse transcriptase uses RNA template for cDNA synthesis
- reverse transcriptase continues to copy chromosomal DNA once it reaches te end of RNA then continues to copy the DNA generated from the staggered break in the target
- second cDNA strand synthesis uses 3’ end as a primer
- repair results in insertion of L1 at target flanked by target site DR
What are SINES?
Non-autonomous retro elements, RNA derived , require active LINE for motility, extremely numerous,
Short interspersed nuclear elements
Give an example of a family of SINES and their properties?
Alu - most numberous in human genome
- related to 7SL RNA
- contain an RNA pol III promoter within sequence, interspersed with non-repetitive DNA
Where are LINES and SINES?
- Individuals with elements in exons will have been subjected to negative selection
- Most in introns, 3’ untranslated regions of genes, heterochromatic regions, intergenic regions
Why are retroelements so inactive?
degenerate due to mutation
host regulatory mechanisms repress them
What are examples of exceptions where LINES have entered genes and disrupted them?
1) 3 separate insertions of LINE element into gene enconding Factor VIII causing haemophilia
2) 11 Alu insertions found cauging diseases including haemophilia B (Factor IX), breast cancer (BRCA2)
How can repetitive elements have an impact on genome evolution?
- unequal crossing over between two repeated elements e.g. L1 flanking a gene can result in genome duplication e.g. in human globin genes
- > divergence of duplicate genes can lead to specialised functions increasing fitness
- unequal crossing over could result un duplication of exons , leading to evolution of proteins with multiple copes of similar exons e.g. fibronectin
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Lecture 1 - genetic variation vs. genome stability23
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Lecture 2 - Examples of PMS33
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Lecture 3 - CO and mating type switch32
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Lecture 8 - replication forks and BIR30
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Lecture 9 - Telomeres and telomerase39
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Lecture 10 - DNA repair systems and SOS37
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Lecture 11 - Mutant genes22
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Lecture 12 - reverse genetics and genome editing29
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Lecture 4 - Site specific recombination and transposition38
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Lecture 5 - Retroelements24
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Lecture 6 - Site specific recombination37
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Lecture 7 - Applications of genome engineering tools0
