1. Bacterial Transposons Author Meenakshi Agarwal, Mehta Gunjan Mentor Dr. Santanu Ghosh

2. Master Layout (Part 1) 5 3 2 4 1 This animation consists of 3 parts: Part 1 – Definition of transposons and keywords Part 2 – Types of transposons Part 3 – Mechanism of transposition

3. 5 3 2 4 1 Part1- Definition of Transposons Alternate nomenclature: 1) Transposable elements 2) Jumping genes 3) Mobile DNA Discovery: First discovered by Barbara McClintock in 1950’s. Definition: Capable of independently replicating itself and inserting the copy into a new position within the same or another chromosome or plasmid, a process called transposition.

4. 5 3 2 4 1 Keywords 1) IS (Insertion sequence) elements: is a small bacterial transposon that carries only the genes needed for its own transposition. 2) Inverted terminal repeats: are the short related or identical sequences present in reverse orientation at the ends of some transposons. 3) Direct repeats: are identical (or closely related) sequences present in two or more copies in the same orientation in the same molecule of DNA.

5. 5 3 2 4 1 Keywords 4) Transposase: an enzyme that binds to ends of transposon and catalyses the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. 5) Resolvase: is the enzyme activity involved in site-specific recombination between two transposons present as direct repeats in a cointegrate structure.

6. 1 5 3 2 4 Part 2-Types of bacterial transposons There are three types of bacterial tranposons: 1) IS elements 2) Composite transposon 3) Non-composite transposon

7. 1 5 3 2 4 IS elements IS (Insertion sequence) elements: The simplest transposons, are autonomous units, each of which codes only for the proteins needed to sponsor its own transposition. Inverted repeats + transposase genes

8. Step 1: 1 5 4 Audio Narration (if any) ‏ DT1 Text to be displayed (if any) ‏ (DT) ‏ Description of the action/ interactivity Structure of IS elements 2 3 Transposase protein 1 st yellow box will appear with lable. 2) Arrow from box and transposase Protein structure will appear. 3)Two red boxes(IR) will appear on both ends. 4) sequences with blue boxes will appear. 5) in last Insertion sequence, IS1 (text)will appear. IS elements are simplest Transposons and contains Inverted repeats and tranposase genes, codes proteins for transposition protein

9. 1 5 3 2 4 Composite transposons Composite transposons: Composite genetic elements are larger than IS elements and contain one or more protein- coding genes in addition to those required for transposition e.g. Tn5, Tn9, Tn10. Two IS elements + antibiotic resistance gene(s).

10. 1 5 4 Audio Narration (if any) ‏ DT1 Text to be displayed (if any) ‏ (DT) ‏ Description of the action/ interactivity Structure of Composite transposons 2 3 1 st Black box will appear with text and lable. 2) Red and yellow boxes at both ends With lables. 3) triangle shaped arrow and text inverted IS will appear antibiotic resistance genes Inverted Repeats Genes for Transposition Inverted IS Structural genes Inverted Repeats These transposons composed of two insertion sequence, which codes genes for transposition, flanking structural genes which codes for various proteins and enzymes i.e. viral or antibiotic resistance

11. 1 5 3 2 4 Non composite transposons Non-composite transposons: Non-composite mobile genetic elements are those which lack IS elements on its ends e.g. Tn3 and Tn7. Inverted repeats + transposase gene + antibiotic resistance gene(s).

12. 1 5 4 Audio Narration (if any) ‏ DT1 Text to be displayed (if any) ‏ (DT) ‏ Description of the action/ interactivity Structure of Non-composite transposons 2 3 1 st blue box with label transposase (tnpA) will appear. 2) Reolvase with tnpB will blow up 3) B –lactamase (bla) will blow up. 4) two yellow boxes at both ends with their lable will appear. 5) arrow with size 4957bp will appear This is the example of Non- composite transposon- Tn3.These transposons encode transposition proteins, have inverted repeats (but no ISs) at their ends. In addition to resistance and virulence genes they may encode catabolic enzymes

13. 1 5 3 2 4 Part 3- Mechanism All transposons use a common mechanism in which staggered nicks are made in target DNA, the transposon is joined to the protruding ends, and the gaps are filled. The order of events and exact nature of the connections between transposon and target DNA determine whether transposition is replicative or nonreplicative.

14. 1 5 3 2 4 Replicative Transposon Replicative transposon is first replicated and then one of the copy will move to the another location in the genome. Thus, the transposon will remain on its original position. “Copy and Paste” Replicative transposition involves two types of enzymatic activity: Transposase that acts on the ends of the original transposon; and Resolvase that acts on the duplicated copies. A group of transposons related to TnA move only by replicative transposition.

15. 1 5 3 2 4 Replicative Transposon Audio Narration (if any) ‏ DT1 Text to be displayed (if any) ‏ (DT) ‏ Description of the action/ interactivity First donor and recipient DNA will appear. Dark pink region from donor molecule will be replicated and it will be inserted at the target site in the recipient molecule. In replicative transposition, the transposon is replicated first from the donor molecule and then it is inserted at the target site present in the recipient molecule.

16. 1 5 3 2 4 Replicative Transposon Replicative transposition occurs through a cointegrate formation. A cointegrate structure is produced by fusion of two replicons, one originally possessing a transposon, the other lacking it; the cointegrate has copies of the transposon present at both junctions of the replicons, oriented as direct repeats. Resolution occurs by a homologous recombination reaction between the two copies of the transposon in a cointegrate. The reaction generates the donor and target replicons, each with a copy of the transposon. Resolvase is the enzyme activity involved in site-specific recombination between two transposons present as direct repeats in a cointegrate structure.

17. 1 5 3 2 4 Replicative Transposon The reactions involved in generating a cointegrate have been defined in detail for phage Mu. The process starts with the formation of the strand transfer complex (sometimes also called a crossover complex). The donor and target strands are ligated so that each end of the transposon sequence is joined to one of the protruding single strands generated at the target site. The crossover structure contains a single-stranded region at each of the staggered ends. These regions are pseudoreplication forks that provide a template for DNA synthesis. (Use of the ends as primers for replication implies that the strand breakage must occur with a polarity that generates a 3 ′ –OH terminus at this point.)

18. 1 5 3 2 4 Replicative Transposon Audio Narration (if any) ‏ DT1 Text to be displayed (if any) ‏ (DT) ‏ Description of the action/ interactivity As shown in figure, one by one each step will appear along with text. Replicative transposition occurs through cointegrate formation. Nick formation occurs on donor and recipient molecule. The single stranded ends of transposon will be ligated to the single stranded ends of recipient molecule. The remaining single stranded portion is replicated using the 3’- OH end as a primer. Thus, the cointegrate structure is formed in which two copies of the transposons are present at the junction between donor and recipient molecules. Resolvase will carry out site- specific recombination between the direct repeats present on the transposons in a cointegrate structure.

19. 1 5 3 2 4 Non-replicative Transposon Non-Replicative transposon leaves its original place and move to the another location in the genome. “Cut and Paste” This type of mechanism requires only a transposase. The insertion elements and composite transposons like Tn5 and Tn10 use this mechanism. Non-replicative transposons leave a break in the donor molecule which is lethal to the cell unless it is repaired.

20. 1 5 3 2 4 Non-replicative Transposon Audio Narration (if any) ‏ DT1 Text to be displayed (if any) ‏ (DT) ‏ Description of the action/ interactivity Donor and recipient molecules will appear first. Transposon (Dark Pink) will be removed from the donor molecule and will be place at the target site in the recipient molecule. In case of Non-replicative transposition, transposon removes from the donor molecule and it integrates into the recipient molecule. The break generated in the donor molecule can be lethal if it is not repaired.

21. Questionnaire 1 5 2 4 3 1.Transposons are a.sequences of DNA that move to different positions within the genome of a single cell. b.sequences of RNA that move to different positions within a cell. c.sequences of DNA that move from one position to another within two cells. 2. Tn 10 is a a.non composite transposon b. a composite transposon c.an mobile genetic element 3.Transposition is of type a.replicative only b.non replicative only c.replicative and non replicative 4. Mobile genetic elements found in bacteria include a.TMV,TMV,DHFR b. PCR, DNA,RNA c.Tn 3,Is 10,Tn 10 5.In replicative transposition a. transposable element is duplicated during the reaction, so that the transposing entity is a copy of the original element. b.Transposition of transposable elements occurs by, "cut-and-paste" mechanism. c.none of the above.

22. Answers to the set of questions 1 a 2 b 3 c 4 c 5 a

23. Links for further reading Books GENES VIII- Benjamin Lewin Principles of GENETICS – 8 th edition Microbial genetics- David Freifelder

24. Transposons are sequences of DNA that can move around to different positions within the genome of a single cell. Three types of bacterial transposons 1) IS elements, 2) Composite and 3) Non-composite. Transposons involves three modes of transposition 1) Replicative, 2) Non- replicative and 3) Conservative Summary