Transposition and transposable elements. Transposable elements “mobile genetic elements”...
-
date post
21-Dec-2015 -
Category
Documents
-
view
234 -
download
3
Transcript of Transposition and transposable elements. Transposable elements “mobile genetic elements”...
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
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
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
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
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”
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
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
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
Target-primed reverse transcription
Explains: Insertions are often 5’ truncatedTransduction of 3’ markers
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
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