BACTERIAL TRANSPOSONS

1. BACTERIAL TRANSPOSONS Miss Hradhaya C Bhatlawande

2. Definitions and Keywords • Transposons - are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. • 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. • IS elements -A short DNA sequence that acts as a simple transposable element

3. Definitions and Keywords DNA polymerase-A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA ligase is a special type of ligase that can link together two DNA strands that have double-strand break a break in both complementary strands of DNA

4. Bacterial Transposons • Bacteria contain two types of transposons • 1]Composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition. • 2]Non composite mobile genetic elements are those which lack IS elements on its ends e.g. is Tn3

5. Composite Transposon • A composite transposon, is flanked by two separate IS elements which may or may not be exact replicas. Instead of each IS element moving separately, the entire length of DNA spanning from one IS element to the other is transposed as one complete unit. IR IR

6. Non composite Transposon • Non-composite transposons (which lack flanking insertion sequences). In each case, transposition requires specific DNA sequences located at the ends (IS1, IS3, Tn5, Tn10, and Tn3) or a multisubunit complex (e.g. Tn7). • Encode transposition proteins, have inverted repeats (but no ISs) at their ends. In addition to resistance and virulence genes they may encode catabolic enzymes

7. Mechanism of transposition • There are two mechanisms of transposition replicative and nonreplicative • During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows, • So the entire transposon is moved from the donor DNA (e.g., a plasmid). • A DNA polymerase fills in the resulting gaps from the sticky ends and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase). The duplications at the target site can result in gene duplication and this is supposed to play an important role in evolution. • Composite transposons will also often carry one or more genes conferring antibiotic resistance

8. Mechanism of transposition(contd) • The conservative mechanism, also called the “cut-and-paste” mechanism, is used by elements like Tn10 . • The element is excised cleanly by double-strand cleavages from the donor DNA • and inserted, with limited repair, between a pair of staggered nicks at the target DNA. • Replicative transposition is a mechanism of transposition in molecular biology in which the transposable element is duplicated during the reaction, so that the transposing entity is a copy of the original element. Replicative transposition is characteristic to retrotransposons and occurs from time to time in class II transposons. • Retrieved from "http://en.wikipedia.org/wiki/Replicative_transposition

9. General mechanism of Transposition Production of protein (enzyme transposase) from the site of transposase(right corner an Tn 5) should be shown.{the site in upper diagram in between IR of IS element.} Action/Motion-Production of protein (enzyme transposase) from the site of transposase (right corner an Tn 5) should be shown

10. Replicative Transposition Single stranded cuts are made on either side of the Transposon and on the opposite sides of the target of the recipient.

11. get This produces 4 free ends in each DNA molecule Two of the ends from the donor are ligated to 2 of the ends of target. This links the two molecules with a single molecule of transposon.

12. The two remaining free 3’ ends are used as primers for DNA polymerase which uses the Transposon DNA as the template.This replicates the transposon and leaves the cointegrate.

14. Nicking Single strranded cuts produce staggered ends in both transposon and target

15. Crossover structure (strand transfer complex) Nicked ends of Transposon are joined to nicked ends of target.

16. Replication from free 3’ end generate cointegrate Single molecule has two types of transposon.

17. Cointegrate drawn as continuous path shows that transposons are at junctions between replicons.

18. NON REPLICATIVE TRANSPOSON

19. First, the transposase makes a double-stranded cut in the donor DNA at the ends of the transposon and makes a staggered cut in the recipient DNA.

20. Each end of the donor DNA is then joined to an overhanging end of the recipient DNA.

21. DNA polymerase fills in the short, overhanging sequences, resulting in a short, direct repeat on each side of the transposon insertion in the recipient DNA.

22. INSTRUCTIONS SLIDE Questionnaire to test the user 1 • Q1]Define tranposition? • Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. • Q2]Give examples of non composite transposons. • IS1, IS3, Tn5, Tn10, and Tn3) or a multisubunit complex (e.g. Tn7)‏ • Q3]Describe the general structure of bacterial transposons. • Ans 2 3 4 5

23. This transposon consists of a chloramphenicol-resistance gene (dark blue) flanked by two copies of IS1 (orange), one of the smallest IS elements. Other copies of IS1, without the drug-resistance gene, are located elsewhere in the E. coli chromosome. The internal inverted repeats of IS1 abutting the resistance gene are so mutated that transposase does not recognize them. During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows, so the entire transposon is moved from the donor DNA (e.g., a plasmid). The target-site sequence at the point of insertion becomes duplicated on either side of the transposon during transposition, which occurs via the replicative mechanism. Note that the 5-bp target-site direct repeat (light blue) is not to scale

24. Q4]Explain the mobile genetic elements found in bacteria. ANS:- Three of the many types of mobile genetic elements found in bacteria. Each of these DNA elements contains a gene that encodes a transposase, an enzyme that conducts at least some of the DNA breakage and joining reactions needed for the element to move. Each mobile element also carries short DNA sequences (indicated in red) that are recognized only by the transposase encoded by that element and are necessary for movement of the element. In addition, two of the three mobile elements shown carry genes that encode enzymes that inactivate the antibiotics ampicillin (ampR) and tetracycline (tetR). The transposable element Tn10, shown in the bottom diagram, is thought to have evolved from the chance landing of two short mobile elements on either side of a tetracyclin-resistance gene; the wide use of tetracycline as an antibiotic has aided the spread of this gene through bacterial populations. The three mobile elements shown are all examples of DNA-only transposons

25. Q5]Illustrate the mechanism of transposition in transposons. • ANS:-

26. Links for further reading 1 Molecular Cell BiolOGY Baltimore -molecUlar biology of the gene watson -Genes Lewin -VOET AND VOET -LEHNINGER -COOPER 2 3 4 5

27. Thank you