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微生物遺傳與生物技術 (Microbial Genetics and Biotechnology) 金門大學 食品科學系 何國傑 教授 Autonomously replicating genetic entities (3) the transposable elements Transposons ※ Transposons are DNA elements that can hop, or transpose, from one place in DNA to another. They are also called “jumping genes”. They carry the enzyme, transposase responsible for transposition, the movement by a transposon. ※ They are discovered by Barbara McClintock in the early 1950s. ※ The transposons now exist in all organisms on the earth, including human. ※ Transposons may offer a way of introducing genes from one bacterium into the chromosome of another bacterium to which it has little DNA sequence homology, so they obviously play an important role in evolution. Transposons ※ Transposition must be tightly regulated and occur only rarely; otherwise, the cellular DNA would become riddled with the transposons, which would have many deleterious effects. In fact, the frequency of transposition varies from once in every 103 to once in every 108 cell division, depending on the type of transposon. It is not higher than the chance of a gene inactivated by other mutation. Almost half of human genome may be the transposons. ※ Genome – The complete DNA sequence of an organism. 人類基因體計畫 人類的染色體為23對,其一半即為構成人類 的基因體,約含有3 X 109鹼基對(bp),其 大小約為大腸桿茵(4.2 X 106 bp)的1,000 倍,這是否表示人類的基因體所含的基因數 目為大腸桿茵的1,000倍(大腸桿茵有2,000 個基因)。答案是否定的,人類基因體計畫 的完成顯示人類的基大約有35,000個。所以 人類基因體含有大量的〞廢物DNA(junk DNA)〞,約佔人類總DNA的97%,這些DNA包 括基因中的隱子,基因間的重覆序列及所謂 的跳躍基因。 The largest of component of the human genome consists of transposons. Other repetitive sequences include large duplications and simple repeats. 22% Overview of transposition I. Types of bacterial tansposons 1. Insertion sequence (IS) elements (1) These transposons are usually only about 750 ~ 2000 bp long and encode little more than the transposae that promote they transposition. (2) Repeats at ends, usually inverted repeats (shown by arrows). (3) IS3 consists of two open reading frames (ORFA and ORFB). (4) ORFB is shifted -1 relative to ORFA, but a programmed -1 (2) Fig.9.2 is the structure of the IS3 element, which contains frameshift causes the synthesis of a fusion protein, ORFAB, which is the active transposase. (5) Smaller protein made from ORFA when the frameshift does not occur regulates transcription of transposase. (6) The target site sequence that is duplicated on the insertion of IS3 is 3 bp long (ex., ATT). The length of such direct repeats is characteristic of each type of transposon. Structure of the insertion sequence element IS3 and its related family member 1. The inverted repeats are shown as arrows, and the 3-bp target sequence that is duplicated after transposition is boxed. 2. OFRA and OFRB encode the N terminus and C terminus of the transposase, which are translated in different reading frames and are not active by themselves. A programmed -1 frameshift put both ORFA and ORFB in the same frame and makes the active transposase The C terminus of the IS3 transposase contains the DDE motif characteristic of this type of transposase. I. Types of bacterial tansposons 2. Composite transposons (1) A larger transposon: Two IS elements of the same type bracket other genes, usually the antibiotic resistant gene(s). (2) Transposition * Each IS element of the same transposon can transpose independently as long as the transposase acts on both of its ends. * Two IS elements are often not completely autonomous, because the active transposase of one IS element can act on the outside ends to promote transposition of the composite transposon when the transposase of the other element is inactive due to a mutation. I. Types of bacterial tansposons 2. Composite transposons i. Outside-end transposition When a transposase acts on the inverted repeats at the farthest ends of a composite transposon, the two IS elements transpose as a unit along with the genes between them. ii. Inside-end transposition A transposase encoded by one IS element can also act on the inside ends of both IS elements. Structures of some composite transposons 1. The active transposase gene is in one of the two IS elements. 2. The IS elements can be in either the same or opposite orientation (arrows). Two IS elements can transpose any DNA between them Either the outside or inside ends of the IS elements in a composite transposon can be used for transposition (a) Transposition with the outside ends of IS10 element would move Tn10, with the tetracycline resistance gene (Tetr) to another DNA. (b) Transposition from the inside ends would create a new composite transposon carrying the Ampr gene and the plasmid origin of replication (Ori) to another DNA. (b)If this new composite transposon hops into a target DNA that does not have a functional origin of replication, it may confer on that DNA the ability to replicate. Rearrangements of DNA caused by composite transposons through inside-ends transposition The neighboring sequences between The original site of insertion of the transposon and the site into which it is trying to transpose will be either deleted or inverted. (B, C, D) 1. If the inside ends (i and ii) do not cross over each other before they attach (join), the neighboring sequences will be deleted. 2. If the inside ends cross over each other before they attach (join), the neighboring sequences will be inverted. 3. The DNA between the two IS elements in the composite transposon will also be deleted. Rearrangements of DNA caused by composite transposons through inside-ends transposition 4. Methods have been developed to select tet-sensitive derivatives of E. coli haerboring the Tn10 transposon. Most of these tet-sensitive derivatives have deletions or inversions of DNA next to the site of insertion of Tn10 element. 5. Inside-end transposition is presumably responsible for most of the often-observed instability of DNA (rearrangement) caused by composite transposon. 6. Some composite transposons have mechanisms to avoid inside-end transposition. Ex., adenines of inside-ends of Tn5 are methylated so that they are recognized less well by the transposase. 7. Assembly of plasmids by IS elements - Many of the resistance gene on plasmids are bracketed by the same IS element. Apparently, the plasmid was assembled in nature by resistance genes hopping onto the plasmid from some other DNA via the bracketing IS elements. Rearrangements of DNA caused by composite transposons through inside-ends transposition 7. Assembly of plasmids by IS elements - Many of the resistance gene on plasmids are bracketed by the same IS element. Apparently, the plasmid was assembled in nature by resistance genes hopping onto the plasmid from some other DNA via the bracketing IS elements. III. Mechanisms of transposition IIIa. A molecular model for transposition of Tn3 (A replicative transposition) 1. Breaks are made in the target DNA and at the ends of the transposon, respectively (1 and 2). 2. The 3’ OH ends of the transposon (dots) are ligated to 5’ PO4 ends of the target DNA (3). The insert (3’) shows details of the ends. 3. The free 3’ ends of the target DNA prime replication in both directions over the transposon to form the cointegrate (4). 4. The cointegrate is resolved by recombination promoted by the resolvase TnpR at the res sites (5) 5. Resolution of the cointegrate give rise to two copies of the transposons, one at the former (or donor) site and a new one at the target site. (The A and B in the target DNA illustrate how the target DNA is reversed in the step 3 for ease of drawing.) A molecular model for transposition of Tn3 (A replicative transposition) III. Mechanisms of transposition IIIa. A molecular model for transposition of Tn3 (A replicative transposition) 6. The transposase cuts the target and donor DNAs and promotes ligation of the ends. 7. The resolvase specifically promotes recombination between trhe res elements in cointegrate. 8. Mu phage replicate itself and insert itself around the chromosome of its bacterial host by a mechanism similar to Tn3. (1) It does not resolve the cointegrate and soon the chromosome becomes riddled with Mu genome. (2) These genomes are packaged directly from chromosomal DNA into the phage head, discarding the bacterial chromosomal DNA between the inserted Mu genomes. IIIb. Transposition by Tn10 and Tn5 • Transposition by a cut-and-paste mechanism (also known as conservative mechanism or nonreplicative transposition) • The transposon is moved from one place and inserted into another place. • Transposon produces a short duplication of target DNA at the ends of the transposon. • Donor DNA probably leaves break and is consequently degraded. • There is no cointegrate formation as it does in the replicative mechanism. Cut-and-paste transposition Details of transposition by DDE transposons • All of transposns discussed so far are considered DDE transposons, because their trasposaes all have two aspartates (D) and one glutamate (E) that are essential for their activity. • These acidic amino acids are not next to each other in polypeptide, but they are together in the active center when the protein is folded. • They hold two Mg2+ that participate in the cleavage of DNA during the transposition event. Details of the mechanism of transposition by Tn5 1. Single copies of the transposae (TnP) bind to each end of the transposon, and then bind to each other, bring the two ends of transposon together (synapsis). 2. Transposase bound at one end cuts the DNA at the other end and vice versa to leave 3’ OH ends at each end of transposon. 3. These activated 3’ OH ends attack the phosphodiester bond on the other strand, forming 3’-5’ phosphodiester hairpins. This cuts the transposon out of the donor DNA. Details of the mechanism of transposition by Tn5 4. When the transposase binds to the target DNA, it cuts the two hairpin ends again and the 3’ OH ends attack phosphodiester bonds 9 bp apart in the target DNA, cutting them, and the 5’ phosphate ends in the target DNA are joined to the 3’ OH ends in the transposon, inserting the transposon into the target DNA. 5 .The 9-bp single-stranded gaps on each side of transposon are filled in by DNA polypomerase to make the 9-bp repeats in the target DNA. Details of the mechanism of transposition by Tn5 Details of the mechanism of transposition by Tn7 • The cut and paste transposon Tn7 can be converted into a replicative transposon by a single amino acid change in one subunit of transposase. • Different subunits of transposase make the cuts in the opposite strands of DNA at the ends of transposon. - TnsA cuts at the 5’ and, and TNsB cuts at 3’ end, - They cut the donor DNA only in the presence of the target DNA. • If the TnsA subunit that makes the cut that leaves the 5’ hydroxyl end is altered by a mutation, transposase will cut only the other strand, leaving a free 3’ OH like a replicative transposase. • Apparently, the transposases need only make the appropriate cuts and joinings, and the replication apparatus of the cell does the rest. Details of the mechanism of transposition by Tn7 • The linear Tn7 transposon does not cut itself out of the donor DNA unless the target DNA is already bound to the transposase, so the 5’ ends are not left exposed for long. The difference and similarity between replicative and nonreplicative transposition • The major difference between replicative and nonreplicative transposition 1. Replicative transposase cuts only one strand at the junction. 2. Nonreplicative transposase makes cuts in both strands in the junction. • The similarity between replicative and nonreplicative transposition 1. The cut 5’ ends of the target DNA are joined to the free 3’ ends of the transposon. 2. The free 3’ ends of target DNA are used as primers for replication that proceeds until a free 5’ end in the donor DNA is reached (The only different is whether the replication has to proceed over the entire transposon (replicative ) or only over the short region that is duplicated (cut and paste). IV. General properties of transposons • Target specificity 1 . No transposable element inserts completely randomly into target DNA: Target specificity of some transposons are relaxed and some are stringent. 2. Tn7 transposes with a high frequency into only one site in E. coli DNA, called attTn7, just downstream of the glmS gene. (1) The transposition machinery consists of five proteins: i. TnsA and TnsB – make up the transposase that cuts and joins the DNA strands. ii. Other proteins play ancillary roles: (i) TnsD – may direct the Tn7 to the target sequence, attTn7. It may induce changes representative of triple-stranded structures in the attTn7 site. (ii) TnsC – (i) event directs TnsC to stimulate transposition into the site. IV. General properties of transposons (iii) TnsE – In the absence of TnsD, TnsE stimulates transposition into other site on chromosome. This transposition is inefficient but random. (2) The glmS gene is highly conserved. i. The product of glmS performs an important step in cell wall biosynthesis. ii. The insertion site of Tn7 is downstream the gene, and has no effect on cell only its transcription termination site. • Effects on genes adjacent to the insertion site – could be negative or positive (Tn5 and Tn10 contain promoter near their termini) IV. General properties of transposons • Regulation of transposition – transposition of most transposons occurs rarely because they self- regulate their transposition. The regulatory mechanisms differ greatly: 1. Tn3 – The TnpR protein represses the transcription of the transposase gene (Tnp). 2. Tn10 – transposition occurs primarily just after a replication fork has passed through the element. (1) Newly replicated E. coli DNA is hemimethylated at GATC sites, and it not only activates the transposase promoter but also increases the activity of the transposon ends. (2) The translation of transposase is also repressed by an antisense RNA. 3. Tn5 - Using a truncated transposase version to inhibit the active one. Regulation of transposition • Regulation of Tn5 transposition 1. Two similar IS50 elements flank the antibiotic resistance genes. 2. An N terminally truncated Tnp (transposase) inhibit the active one. 3. Dam methylation of the inside ends (IEs) of the IS50 prevents the transposase from cutting IEs and transposing the individual IS50 elements. • OE, outer ends; Inh, inhibitor of transposase Tnp; The dashed lines indicate that Tnp and Inh made from IS50L are defective. IV. General properties of transposons • Target immunity – Some transposons prefer not to hop close in the DNA to another transposon of the same type. Immunity can extend over 100,000 bp of DNA. 1. If two transposons were to insert close to each other, would cause large deletions and often lead to the death of the cell. Also, the presence of two transposons close to each other can cause instability in chromosome. 2. Only Mu, Tn3 and Tn7 families of transposons are known to exhibit target immunity. (1) MuB protein seems to be indirectly resposible for the immunity. (2) The binding of MuB to a DNA make it a target for the MuA transposase, which then promotes transposon into DNA. (3) The binding of MuA then cause MuB to dissociate from DNA. (4) Once a transposon has inserted, a copy of MuA may remain bound to the end of the inserted transposon, and prevent the binding of other MUB to the same target DNA and other transposition into that DNA. (5) A similar mechanism may explain target site immunity by Tn7, and the resposible proteins are TnsB and TnsC. IV. General properties of transposons • Target immunity – Some transposons prefer not to hop close in the DNA to another transposon of the same type. • Immunity can extend over 100,000 bp of DNA. 1. If two transposons were to insert close to each other, would cause large deletions and often lead to the death of the cell. 2. Also, the presence of two transposons close to each other can cause instability in chromosome. V.Transposon mutagenesis • Transposons are useful for mutagenesis should have the following properties: 1. It should transpose at a fairly high frequency. 2. It should not be very selective in its target sequence. 3. It should carry an easily selectable gene, such as one for resistance to an antibiotic. 4. It should have a broad host range for transposition if it is to be used in several different kinds of bacteria. Transposon Tn5 mutagenesis Transposon Tn5 mutagenesis • Transposon Tn5, for example in many types of G – bacteria. • There are no equally universal methods for G + bacteris. (A) A standard protocol for transposon mutagenesis of G- bacteria: 1. A suicide ColE1-derived plasmid contains a mob site and transposon Tn5 . 2. The relaxase of this suicide plasmid recognizes the coupling protein of promiscuous plasmid RP4. 3. This suicide plasmid is mobilized into the bacterium by the products of the RP4 transfer genes, which are inserted in the chromosome. 4. Tn5 hops into the chromosome of the recipient cell, and the ColE1 plasmid is lost because it can not replicate. Transposon Tn5 mutagenesis (B) Random transposon mutagenesis of a plasmid 1. Transposon Tn5 is introduced into cells on a suicide vector. 2a. The culture is incubated, allowing the Tn5 time to hop, either into the chromosome (large circle) or into a plasmid (small circle). 2b. Plating on kanamycin-containing medium results in the selection of cells in which a transposition has occurred. 3. Plasmid is prepared from Kanr cells and used to transform Kans cells. 4. Selection for Kanr allows the identification of cells that has acquired a Tn5-carrying plasmid. Cloning genes mutated with a transposn insertion • A transposon used for mutagenesis of a chromosome contains a plasmid origin of replication (ori). • The chromosome is cut with a restriction enzyme, ex., EcoRI which no cut in transposon, and religated. • Transform E. coli with ligated mix, the resulting plasmid in the Ampr transformants will contain the chromosomal sequences that flanked the transposon . Cloning genes mutated with a transposn insertion