Download BACTERIAL TRANSPOSONS

BACTERIAL
TRANSPOSONS
Miss Hradhaya C Bhatlawande
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
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
Bacterial Transposons
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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
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
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
Mechanism of transposition
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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).
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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.
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Composite transposons will also often carry one or more genes conferring
antibiotic resistance
Mechanism of transposition(contd)
• The conservative mechanism, also called the “cut-and-paste” mechanism, is
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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
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
Replicative Transposition
Single stranded cuts are made on
either side of the Transposon and
on the opposite sides of the
target of the recipient.
• 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.
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.
Nicking
Single strranded cuts produce staggered ends in both transposon and target
Crossover structure (strand transfer complex)
Nicked ends of Transposon are joined to nicked ends of target.
Replication from free 3’ end generate cointegrate
Single molecule has two types of transposon.
Cointegrate drawn as continuous path shows that transposons
are at junctions between replicons.
• NON REPLICATIVE TRANSPOSON
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.
Each end of the donor DNA is then
joined to an overhanging
end of the recipient DNA.
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.
INSTRUCTIONS SLIDE
1
Questionnaire to test the user
• Q1]Define tranposition?
are sequences of DNA that can move around to different positions within
2• Transposons
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)
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Q3]Describe the general structure of bacterial transposons.
3• Ans
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• This transposon consists of a chloramphenicolresistance 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
• 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 DNAonly transposons
• Q5]Illustrate the mechanism of
transposition in transposons.
• ANS:-
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Links for further reading
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Molecular Cell BiolOGY
Baltimore
-molecUlar biology of the gene watson
-Genes Lewin
-VOET AND VOET
-LEHNINGER
-COOPER
Thank you