Molecular Biology Fourth Edition Lecture PowerPoint to accompany Molecular Biology Fourth Edition Robert F. Weaver Chapter 23 Transposition Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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

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)

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

Generating Host DNA Direct Repeats

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

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

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”

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

Detailed Tn3 Transposition

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

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

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

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

Transposable Elements in Maize

Structures of Ac and Ds

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

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

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

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

Rearrangement of Antibody Light Chain Gene

Antibody Heavy Chain Coding Regions Human heavy chain is encoded in 48 variable segments 23 diversity segments 6 joining segments 1 constant segment

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

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

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

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

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

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

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

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

Ty Transcription

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

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

Group II Introns Group II introns Group II retrotransposition: 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