Transposition and transposable elements

Transposable elements

• “mobile genetic elements”• comprise 45% of human chromosomal DNA “middle

repetitive DNA” • contribute to spontaneous mutation, genetic

rearrangements, horizontal transfer of genetic material• aid speciation and genomic change (in bacteria

transposons are often associated with antibiotic resistance genes)

• cells must depress transposition to insure genetic stability

Types of transposable elements• DNA vs. RNA

• viral vs. nonviral

• replicative mechanism vs. excision mechanism

transposon

transposoninsertion mutation

Discovery of transposons

• Barbara McClintock 1950’s Ac Ds system in maize influencing kernel colorunstable elementschanging map positionpromote chromosomal breaks

• Rediscovery of bacterial insertion sequencessource of polar mutationsdiscrete change in physical length of DNAinverted repeat ends: form “lollipops” in EM after

denaturation/reannealing

Composite bacterial transposons

• repeated ends, usually inverted, sometimes direct

• repeated ends themselves are IS elements and can independently transpose

• ends mobilize all intervening DNA• often antibiotic resistance genes (examples

Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline)

• often reside on plasmids

Basic minimal insertion sequence structure

ends: genetically required, in cistnp (transposase): genetically required, trans-acting

tnp ORF

Element 1 Element 2 Transposition?

wt wt 1 and 2

ends- ends- neither

trp- tnp- neither

ends- wt only 2

tnp- wt 1 and 2

tnp- ends- only 1

Structure of Tn34957 bp

ampcillin-resistancebla

transposase

tnpA

“repressor”tnpRTn3 resolvase

3 trans-acting genes:

2 cis-acting sites:

38 bp inverted repeat ends120 bp “IRS” or res internal resolution site

tnpR and res mutations cause accumulation of “co-integrate structure”

direct repeat of Tn

cointegrate

2 types of DNA tranposons

• excisive mechanismexamples: Tn5, Tn10, P elements

• replicative mechanismexamples: Tn3, bacteriophage Mu

Replicative transposons

• orignal cut of transposon is only nick and only one strand is initially ligated

• element replicates through itself• produces as intermediate a “co-integrate”

structure • co-integrate is resolved by resolvase (as

TnpR of Tn3) and at specific site (as res of Tn3)

Excisive transposons

• cut-and-paste mechanism• cut themselves out of original site, producing

double strand break• cut target site and ligate to element ends, thereby

inserting at new site• original site break repaired

usually with sister chromosome, restoring transposon at original site

sometimes end healed without transposon, can also be associated with deletion at excision site

Source of target site duplication “TSD”GAC

CTG

CTG

GACStaggered cleavage of target

CTG

GACLigation of transposon DNA

GAC

CTG

Repair replication generates short direct repeats

“degenerate” transposons• many naturally occurring transposable elements have

suffered mutation and are no longer active• some of these may have cis-acting end mutations and cannot

be mobilized• others may have intact ends but no transposase: these can be

mobilized by a element that is tnp+ (“autonomous” element)• Ac Ds system is an example of latter: Ac (activator) can

mobilize Ds (dissociator)• MITEs (minature inverted repeat transposable elements) are

nonautonomous DNA elements• SINEs are retrotransposon version (LINEs)

Comparison of transposition reactions

Direct transesterification reactions DDE motif transposase (integrase)

Comparison of tranposase structural organization

Mechanism of transposases and retroviral integrases

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Classification of retroelements

• Have obligate RNA intermediate, use reverse transcriptase (RT, RNA-dependent DNA polymerase)

• LTR-retroelements: long terminal repeatsTy1/copia, Ty3/gypsy, retroviruses

• Non-LTR-retroelements “retroposons”LINES

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Characteristics of LTR retroelements

• Long terminal repeats: required for replication cycle• Genes: gag, pol, (viruses also have env)• Pol is polyprotein which gives rise to RT (reverse

transcriptase), IN (integrase) RH (RNase H), PR (protease)

• Forms VLPs virus-like particles• Integrase is functionally and structurally similar to

transposase of DNA transposons, DDE motif• Integration gives characteristic TSD

LTR element replicationtRNA primer

Multiple template“jumps”

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Characteristics of non-LTR retroelements

• 2 ORFs, orf1, orf2• Variable TSD• ORF2 gives rise to EN, endonuclease,

(similar to APE) and RT• Uses target primed reverse transcription

TPRT• Can transduce 3’ downstream non-element

segments

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Target-primed reverse transcription

Explains: Insertions are often 5’ truncatedTransduction of 3’ markers

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Human L1 (LINE-1) retroelement

• 15% of human DNA

• 520,000 copies, only 3-5,000 are full-length

• Associated with human disease loci

• Transpose specifically in germ line