Transposons & Mechanisms of Transposition
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Transcript of Transposons & Mechanisms of Transposition
Transposons & Mechanisms of Transposition
Krystine Garcia, Tao Jing, Alexander Meyers
Genomic Distribution98.5% of human genome non-
coding25% non-repeat spacer DNARepetitious DNA
-Tandemly repeated genes-Sattelite DNA (6%)
Interspaced repeats
Transposable Elements
Interspaced repeats have the capability to “move around” in the genome, and are thus referred to as transposable elements.
Transposition in germ cells are passed down to progeny resulting in an accumulation in the genome.
Transposable ElementsBegan as symbiont DNATransposons provide a
mechanism for bringing about DNA rearrangements throughout evolution
Adjacent DNA sequences sometimes mobilized
Transposons, retrotransposons
Transposons and RetrotransposonsTransposons excise themselves
and move to another locationRetrotransposons duplicate and
reintegrate themselves
IS: Insertion Sequence
IS: Insertion Sequence
TransposaseCis-acting enzymeIs coded for in
transposonCuts the IS at
inverted repeatsTransmits it to
another part of the DNA at a target sequence
Multiple types of transposase
http://www.rcsb.org/pdb/101/motm.do?momID=84
Transposase Regulation
Frequency of transposition is regulated by transposase regulation
Not all transposase genes are transcribed
http://www.rcsb.org/pdb/101/motm.do?momID=84
Autonomous & NonautonomousAutonomous (activator elements)
are very similar to bacterial IS elements in structure and function
Nonautonomous (dissociation elements) lack transposase gene◦Cannot move by themselves◦Must have a cis transposable
element with transposase gene to move
3 principle classes of transposons:1. DNA transposons: move using cut and paste or replicative mechanism
2. Virus-like retrotransposons (aka long terminal repeat [LTR] retrotransposons): RNA intermediate, includes retroviruses
3. Poly-A retrotransposons (aka nonviral retrotransposons): RNA intermediate
Cut and paste mechanism of transposition:
Nonreplicative1. Transposase (usually 2 or 4 subunits)
binds terminal inverted repeats2. Brings 2 ends together stable protein
complex called synaptic complex or transpososome
3. Transposase cleaves one DNA strand at each end at junction between transposon DNA and host DNA transposon sequence terminates with free 3’-OH groups at each end
4. Other DNA strands cut by various mechanisms transposon excised
Cut and paste mechanism of transposition:
5. 3’-OH ends of transposon DNA attack DNA phosphodiester bonds at site of new insertion (target DNA)
6. Nicks introduced in other target DNA strands few nucleotides apart transposon joined via reaction called DNA strand transfer
7. Few nucleotides between nicks leaves small ss gaps filled in by host DNA repair polymerase small target site duplications on either side transposon
8. DNA ligase seals final nicks9. Ds break where transposon left
repaired by homologous recombination
Nontransferred strand cleavage:Nontransferred strands = 5’ ends of transposon (ends not
covalently linked to target DNA during strand transfer)
Cleavage can use enzyme other than transposase:◦ Tn7 encodes specific protein called TnsA with structure similar
to restriction enzyme – works with transposase to cleave nontransferred strands
Cleavage can be performed by transposase:◦ Tn5 and Tn10 form DNA hairpin (3’-OH attacks its
complementing strand) to cause nicks hairpin opened by transposase 3’-OH’s join to target DNA via strand transfer
◦ Hermes forms DNA hairpins in target DNA
Nontransferred strand cleavage:
Replicative transposition:Transposon DNA replicated during
each round of transposition1. Transposase assembles on each end of
transposon to form transpososome2. Transposase introduces nicks at
junctions between transposon and flanking host DNA generates 3’-OH ends on transposon (but transposon NOT excised from flanking DNA)
3. 3’-OH joined to target DNA by strand transfer reaction (same mechanism as cut-and-paste) intermediate is double branched DNA molecule
Replicative transposition:4. 3’ ends transposon covalenty linked to
target DNA, but 5’ ends still linked to old flanking DNA
5. 2 branches like replication forks, DNA replication proteins assemble at these forks, 3’-OH serves as primer
6. Replication proceeds through transposon and stops at 2nd fork 2 copies of transposon flanked by short target site duplications
Frequently causes chromosomal inversions and deletions detrimental to host
Virus-like retrotransposons and retroviruses:
Carry inverted terminal repeats (recombination sequences) embedded within longer direct repeat sequences (aka long terminal repeats [LTR])
Encode 2 proteins needed for mobility: integrase (transposase) and reverse transcriptase
Virus-like retrotransposons and retroviruses:
Reverse transcriptase: Enzyme that uses RNA template to synthesize DNA
Retrovirus: genome packaged into viral particle, leaves host cell, infects new cell
Retrotransposon: can only move to new DNA sites within cell
Virus-like retrotransposons and retroviruses:1. Retrotransposon DNA transcribed into
RNA by host RNAP (transcription starts at promoter within LTR)
2. RNA reverse-transcribed (by RT) RNA:DNA dsDNA (cDNA)
3. Integrase (transposase) recognizes and binds ends of cDNA then cleaves few nucleotides off 3’ end of each strand (just like cleavage step of DNA transposons)
4. Integrase performs strand transfer reaction to insert 3’ ends into target DNA
5. Gap fill and ligation by host proteins
Transposon Excision
Plant genomes are rich in transposons:
Barbara McClintock discovered transposons in the late 1940’s
Maize color varigation due to chromosome breakage by transposition
Snapdragons: size of white patches related to frequency of transposition