Lecture 5: Transposition mechanisms, CSSR Flashcards
Nonretrovrial retrotransposase structure
Poly A at 3’ end of RNA transcript; 5’ end is often truncated
If DNA sites are in same orientation for CSSR:
DNA can be integrated or excised
Difference of CSSR and transposition
CSSR need special sites on EACH DNA that serve as recognition sites for recombinase
Form transient high energy covalent bonds and use it to complete DNA rearrangement
Step 1/2 of non retroviral retransposition
L1 RNA synthesis
Synthesis of reverse transcriptase/endonuclease
Step 1 of retroviral like transposon
Entire transposon transcribed by host
RNA transcript transcribed contains reverse transcriptase enzyme
Non retroviral transposon characteristics
Comprise 40% of human genome
Most are immobile
Require endonuclease and reverse transcriptase to move, which they must use from other transposons
Retroviral-like retrotransposon method of movement
Moves via an RNA intermediate whose production is driven by promotor in the LTR
Retroviral-like transposon enzymes for movement
Reverse transcriptase
Integrase
DNA-only transposon moves how
Moves as DNA, either by cut-and-paste or replicative pathways
How does central intermediate attack target
Creates staggered break (breaking two phosphodiester bonds)
Breaks are repaired and result in duplication of target DNAs insertion site
Nonretroviral retrotransposon enzymes required
Reverse transcriptase
Endonuclease
Nonretroviral retrotransposon method of movement
Moves via RNA intermediate that is often synthesized from a neighboring promotor
Transpososome
DNA only transposon
Two transposase molecules come together forming a loop juxtaposing two ends of element
If DNA sites are in opposite orientation for CSSR:
DNA sequence is inverted instead of excised
Transposase is what, acts on what, allows what
Enzyme encoded by transposon itself (DNA-only transposon)
Acts on specific DNA sequence on each end of transposon
Allows insertion into target DNA site
Conservative site specific recombination does what
Mediates rearrangements of other types of mobile DNA elements
DNA only transposon structure
Short inverted repeats at each end
Step 4 of non retroviral retransposition
Endonuclease nicks the target DNA, revealing 3’ OH to serve as primer in reverse transcription step
Transposons can provide and produce what
Provide benefits to the cell (abx resistance in bacteria)
Produce genetic variation
“Hole” in donor chromosome on transposon is repaired by which three mechanisms and what is the result of each
DS break repair (transposon restored) Homologous recombination (not restored) Non homologous end joining (creates mutation at break site)
Retroviral-like retrotransposon structure
Directly repeated long terminal repeats (LTRs) at each end
Difference in outcome of CSSR depends on
The relative orientation of DNA sites
Step 2 of retroviral like transposon
Reverse transcriptase makes DS DNA copy of RNA molecule via DNA/RNA intermediate
Central intermediate
DNA-only transposon
Formed after transposase cuts the loop formed by two transposase monomers which recognized end sequences and brought them together
Transposons are
Mobile genetic elements “jumping genes”.
Can move from one position in genome to another
Integrase function
Cuts one strand at each end of viral sequence, exposing 3’ ends which attack phosphodiester bonds on target DNA
Step 3 of retroviral like transposon
DS DNA integrates into site on chromosome using integrase
Step 3 of non retroviral retransposition
Endonuclease and reverse transcriptase bind L1 RNA
Step 6 of non retroviral retransposition
Double stranded L1 copy inserted into target DNA
Step 5 of non retroviral retransposition
Single stranded DNA copy of L1 directly linked, DNA primed reverse transcription creates double stranded L!
Different results of CSSR
DNA integration
DNA excision
Inversion
DNA-only transposon- what is required for movement
Transposase
Sequences recognized by transposase necessary for movement
