Molecular BiologyFourth Edition

Chapter 23

Transposition

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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23.1 Bacterial Transposons

• A transposable element moves from one DNA address to another

• Originally discovered in maize, transposons have been found in all kinds of organisms– Bacteria– Plants– Humans

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Discovery of Bacterial Transposons

• Phage coat is made of protein

• Always has the same volume

• DNA is much denser than protein

• More DNA in phage, denser phage

• Extra DNAs that can inactivate a gene by inserting into it were the first transposons discovered in bacteria

• These transposons are called insertion sequences (ISs)

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Insertion Sequences

• Insertion sequences are the simplest type of bacterial transposon

• They contain only the elements necessary for their own transposition– Short inverted repeats at their ends– At least 2 genes coding for an enzyme, transposase

that carries out transposition

• Transposition involves:– Duplication of a short sequence in the target DNA– One copy of this sequence flanks the insertion

sequence on each side after transposition

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Generating Host DNA Direct Repeats

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Complex Transposons

• The term “selfish DNA” implies that insertion sequences and other transposons replicate at the expense of their hosts, providing no value in return

• Some transposons do carry genes that are valuable to their hosts, antibiotic resistance is among most familiar

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Antibiotic Resistance and Transposons

• Donor plasmid has Kanr, harboring transposon Tn3 with Ampr

• Target plasmid has Tetr

• After transposition, Tn3 has replicated and there is a copy in target plasmid

• Target plasmid now confers both Ampr, Tetr

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Transposition Mechanisms• Transposons are sometimes called “jumping

genes”, DNA doesn’t always leave one place for another

• When it does, nonreplicative transposition– “Cut and paste”– Both strands of original DNA move together from 1

place to another without replicating

• Transposition frequently involves DNA replication– 1 copy remains at original site – New copy inserts at the new site– Replicative transposition– “Copy and paste”

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Replicative Transposition of Tn3

• In first step, 2 plasmids fuse, phage replication, forms a cointegrate – coupled through pair of Tn3 copies

• Next is resolution of cointegrate, breaks down into 2 independent plasmids, catalyzed by resolvase gene product

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Detailed Tn3 Transposition

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Nonreplicative Transposition

• Starts with same 2 first steps as in replicative transposition

• New nicks occur at arrow marks

• Nicks liberate donor plasmid minus the transposon

• Filling gaps and sealing nicks completes target plasmid and its new transposon

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23.2 Eukaryotic Transposons

• Transposons have powerful selective forces on their side

• Transposons carry genes that are an advantage to their hosts– Their host can multiply at the expense of

completing organisms– Can multiply the transposons along with rest

of their DNA

• If transposons do not have host advantage, can replicate themselves within their hosts

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Examples of Transposable Elements

• Variegation in the color of maize kernels is caused by multiple reversions of an unstable mutation in the C locus, responsible for kernel color

• Mutation and its reversion result from Ds (dissociation) element– Transposes into the C gene– Mutates it– Transposes out again, revert to wild type

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Ds and Ac of Maize

• Ds cannot transpose on its own

• Must have help from an autonomous transposon, Ac (for activator)– Ac supplies transposase– Ds is an Ac element with most of its middle

removed– Ds needs

• A pair of inverted terminal repeats• Adjacent short sequences that Ac transposase can

recognize

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Transposable Elements in Maize

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Structures of Ac and Ds

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P Elements

• The P-M system of hybrid dysgenesis in Drosophila is caused by conjunction of 2 factors:– Transposable element (P) contributed by the male– M cytoplasm contributed by the female allows

transposition of the P element

• Hybrid offspring of P males and M females suffer multiple transpositions of P element

• Damaging chromosomal mutations are caused that render the hybrids sterile

• P elements have practical value as mutagenic and transforming agents in genetic experiments with Drosophila

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23.3 Rearrangement of Immunoglobulin Genes

• Mammalian genes use a process that closely resembles transposition for:– B cell antibodies– T cell receptors

• Recombinases involved in these processes have similar structures

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Antibody Structure

• Antibody is composed of 4 polypeptides– 2 heavy chains– 2 light chains

• Sites called variable regions – Vary from 1 antibody

to another– Gives proteins their

specificity

• Rest of protein is constant region

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Immune System Diversity

• Enormous diversity of immune system is generated by 3 basic mechanisms:– Assembling genes for antibody light chains

and heavy chains from 2 or 3 component parts

– Joining the gene parts by an imprecise mechanism that can delete bases or add extra bases

– Causing a high rate of somatic mutations, probably during proliferation of a clone if immune cells

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Rearrangement of Antibody Light Chain Gene

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Antibody Heavy Chain Coding Regions

Human heavy chain is encoded in – 48 variable segments– 23 diversity segments– 6 joining segments– 1 constant segment

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Recombination Signals

• The recombination signal sequences (RSSs) in V(D)J recombination consist of:– Heptamer– Nonamer – Separated by 12-bp or 23-bp spacers

• Recombination occurs only between a 12 signal and a 23 signal

• Guarantees that only 1 of each coding region is incorporated into the rearranged gene

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The Recombinase

• Recombination-activating gene (RAG-1) stimulated V(D)J joining activity in vivo

• Another gene tightly liked to RAG-1 also works in V(D)J joining, RAG-2

• These genes, RAG-1 and RAG-2, are expressed only in pre-B and pre-T cells

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Mechanism of V(D)J Recombination

• RAG1 and RAG2 introduce single-strand nicks into DNA adjacent to either a 12 signal or 23 signal

• Results in transesterification where newly created 3’-OH group: – Attacks the opposite strand– Breaks it– Forms hairpin at the end of the coding segment

• Hairpins then break in an imprecise way that allows joining of coding regions with loss of bases or gain of extra bases

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23.4 Retrotransposons

• Retrotransposons replicate through an RNA intermediate

• Retrotransposons resemble retroviruses– Retroviruses can cause tumors in vertebrates– Some retroviruses cause diseases such as

AIDS

• Before studying retrotransposons, look at replication of the retroviruses

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Retroviruses

• Class of virus is named for its ability to make a DNA copy of its RNA genome

• This reaction is the reverse of the transcription reaction – reverse transcription

• Virus particles contain an enzyme that catalyzes reverse transcription reaction

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Retrovirus Replication

• Viral genome is RNA, with long terminal repeats at each end

• Reverse transcriptase makes linear, ds-DNA copy of RNA

• ds-DNA copy integrates back into host DNA = provirus

• Host RNA polymerase II transcribes the provirus to genomic RNA

• Viral RNA packaged into a virus particle

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Model for Synthesis of Provirus DNA

• RNase H degrades the RNA parts of RNA-DNA hybrids created during the replication process

• Host tRNA serves as primer for reverse transcriptase

• Finished ds-DNA copy of viral RNA is then inserted into the host genome

• It can be transcribed by host polymerase II

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Retrotransposons

• Several eukaryotic transposons transpose in a way similar to retroviruses– Ty of yeast– copia of Drosophila

• Start with DNA in the host genome– Make an RNA copy– Reverse transcribe it within a virus-like particle into

DNA that can insert into new location

• HERVs likely transposed in the same way until ability to transpose lost– HERV = human endogenous retroviruses

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Ty Transcription

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Non-LTR Retrotransposons• LTR are lacking in most retrotransposons• Most abundant type lacking LTR are LINEs and

LINE-like elements– Long interspersed elements– Encode an endonuclease that nicks target DNA– Takes advantage of new DNA 3’-end to prime reverse

transcriptase of element RNA– After 2nd strand synthesis, element has been

replicated at target site

• New round of transposition begins when the LINE is transcribed

• LINE polyadenylation signal is weak, so transcription of a LINE often includes exons of downstream host DNA

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Nonautonomous Retrotransposons

• Nonautonomous retrotransposons include very abundant human Alu elements and similar elements in other vertebrates

• Cannot transpose by themselves as they do not encode any proteins

• Take advantage of retrotransposition machinery of other elements such as LINE

• Processed pseudogenes likely arose in same manner

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Group II Introns• Group II introns

– Retrohome to intronless copies same gene by:• Insertion of an RNA intron into the gene• Followed by reverse transcription• Then second-strand synthesis

– Retrotranspose by: • Insertion of an RNA intron into an unrelated gene • Target-primed reverse transcription • Lagging-strand DNA fragments as primers

• Group II retrotransposition: – Forerunner of eukaryotic spliceosomal introns– Accounted for appearance in higher eukaryotes