Dr Moni Kumari

M.Sc 2nd Semster

Transposable elements in prokaryotes

13-8-2020

PG BOTANY DEPARTMENT GAYA COLLEGE, GAYA

DNA transposons have been found in both prokaryotic and eukaryotic organisms.

In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other
genes associated with virulence.

The four transposable genetic elements in prokaryotes are:

(1) Bacterial Insertion Sequences

Insertion Sequences or Insertion-Sequence (IS) Elements:

Insertion sequences, or insertion-sequence (IS) elements, are now known to be segments of
bacterial DNA that can move from one position on a chromosome to a different position on
the same chromosome or on a different chromosome. An IS element contains only genes
required for mobilizing the element and inserting the element into a chromosome at a new
location. Is elements are normal constituents of bacterial chromosome and plasmids.

When IS elements appear in the middle of genes, they interrupt the coding sequence and
inactivate the expression of that gene. Owing to their size and in some cases the presence of
transcription and translation termination signals, IS elements can also block the expression of
other genes in the same operon if those genes are downstream from the promoter of the
operon.

IS elements were first found in E. coli as a result of their affects on the expression of a set of
three genes whose products are needed to metabolize the sugar galactose as a carbon source.

Careful investigations showed that the mutant phenotypes resulted from the insertion of an
approximately 800 base pairs (bp) DNA segment into a gene. This particular DNA segment is
now called insertion sequencel (IS1).

Properties of IS Elements:
Is1 is the genetic element capable of moving around the genome. It integrates into the
chromosome at locations with which it has no homology, thereby distinguishing it from
recombination. This event is an example of transposition event.
There are number of IS elements that have been identified in E. coli, including IS1, IS2, and
IS 10, each present in 0 to 30 copies per genome, and each with a characteristic length and
unique nucleotide sequence.

IS 1 is 768 bp long, and is present in 4 to 19 copies on the E. coli chromosomes. IS2 is

present in 0 to 12 copies on the E. coli chromosome and in one copy on the F plasmid, and IS
10 is found in a class of plasmids called R plasmid that can replicate in E. Coli

Among prokaryotes, the IS elements are normal cell constituents, that is, they are found in
most cells. Altogether, IS elements constitute approx. 0.3% of the cell’s genome. All IS
elements that have been sequenced, end with perfect or nearly perfect inverted terminal
repeats (IRs) of between 9 and 41 bp. This means that essentially the same sequence is found
at each end of an IS but in opposite orientations.

IS Transposition:
When transposition of an IS element takes place, a copy of the IS element inserts into a new
chromosome location while the original IS elements remains in place. That is, transposition
requires the precise replication of the original IS element, using the replication enzymes of
the host cell. The actual transposition also requires an enzyme encoded by the Is element
called transposase.

The IR sequences are essential for the transposition process, that is, those sequences are
recognized by transposase to initiate transposition. Is elements insert into the chromosomes at
sites with which they have no sequence homology?

Genetic recombination between non-homologous sequences is called illegitimate

recombination. The sites into which IS elements insert are called target sites. The process of
IS insertion into a chromosome is shown in Figure below. Firstly, a taggered cut is made in
the target site and the IS element is then inserted, becoming joined to the jingle-stranded
ends.

The gaps are filled in by DNA polymerase and DNA ligase, producing an integrated IS
element with two direct repeats of the target site sequence flanking the IS element. ‘Direct’ in
this case means that the two sequences are repeated in the same orientation. The direct
repeats are called target site duplications. The sizes of target site duplication vary with the IS
elements, but tend to be small. Integration of some IS elements show preference for certain
regions, while others integrate only at particular sequences.

All copies of a given IS element have the same sequence, including that of the inverted
terminal repeats. Mutations that affect the inverted terminal repeat sequence of IS elements
affect transposition, indicating that the inverted terminal repeat sequences are the key
sequences recognized by transposase during a transposition event.

(2) Prokaryotic Transposons

A transposon (Tn) is more complex than an IS elements. A transposon is a mobile DNA

segment that, like an IS element, contains genes for the insertion of the DNA segment into
the chromosome and for the mobilization of the element to other locations on the
chromosome.

There are two types of prokaryotic transposons: composite transposons and non-composite
transposons.
Composite Transposons: They are complex transposons with a central region containing
genes, e.g., drug resistance genes, flanked on both sides by IS elements (also called IS
modules). Composite transposons may be thousands of base pairs long. The IS elements are
both of the same types and are called IS-L (for “left”) and IS-R (for “right”). Depending upon
the transposon, IS-L and IS-R may be in the same or inverted orientation relative to each
other. Because the ISs themselves have terminal inverted repeats, the composite transposons
also have terminal inverted repeats.

The Tn 10 transposon is 9,300 bp long and consists of 6,500 bp of central, nonrepeating DNA
containing the tetracycline resistance gene flanked at each end with a 1,400-bp IS element.
These IS elements are designated IS10L and IS10R and are arranged in an inverted
orientation. Cells containing Tn 10 are resistant to tetracycline resistance gene contained
within the central DNA sequence.

Transposition of composite transposon occurs because of the function of the IS elements they
contain. One or both IS element supplies the transposase. The inverted repeats of the IS
elements at the two ends of the transposon are recognized by transposase to initiate
transposition (as with transposition of IS elements).

Transposition of Tn 10 is rare, occurring once in 10 cell generations. This is the case because
less than one transposase molecule per cell generation is made by Tn 10. Like IS elements,
composite transposons produce target site duplications after transposition.

NON-COMPOSITE: They like composite transposons, contain genes such as those for drug
resistance. Unlike composite transposons, they do not terminate with IS elements. However,
they do have the repeated sequences at their ends that are required for transposition. Tn3 is a
non-composite transposon.

Tn3 has 38 bp inverted terminal repeats and contains three genes in its central region. One of
those genes, bla, encodes β-lactamase which breaks down ampicillin and therefore makes
cells containing Tn3 resistant to ampicillin. The other two genes, tnpA and tnpB, encode the
enzymes transposase and resolvase that are needed for transposition of Tn3 (Fig. 12.4).
Transposase catalyzes insertion of the Tn into new sites, and resolvase is an enzyme involved
in the particular re-combinational events associated with transposition.

Resolvase is not found in all transposons. The genes for transposition are in the central region
for non-composite transposons, while they are in the terminal IS elements for composite
transposons. Non composite transposons also cause target site duplications when they move.

(3) Insertion-Sequence Elements and Transposons in Plasmids

Insertion Sequences (IS-Elements): The insertion sequences are the simplest form
of transposable elements found in prokaryotes. The IS-elements are normal constituents of
the bacterial genome. They may be present in the chromosome or extra-chromosomal
genetic elements, called plasmids.

They were first discovered in connection with genes controlling galactose utilization in E.
coli.

For example, IS-elements present in the F-plasmid of E. coli are involved in the integration of
the F-plasmid with the bacterial chromosome producing Hfr strains. During the process,
genetic exchange between IS-elements of the plasmid and the chromosome takes place.

There are many IS-elements in bacteria which differ in their nucleotide sequence. But all
such elements have some common features in their structure. One important characteristic of
all IS-elements is the presence of 9 to 41 nucleotide-long sequences at each end repeated
nearly identically, but oriented in opposite directions. These sequences are called inverted
terminal repeats. In between the terminal repeats is included a long stretch of DNA double
helix containing several hundred to more than a thousand base pairs.
The central segment of prokaryotic IS-elements carries genetic information for synthesizing
only one or two enzymes which are required for insertion of the element in its target site. One
of these enzymes is transposase. Another enzyme, resolves may also be produced by some
IS-elements. It is a repressor.

The IS-elements may be present in multiple copies in bacterial cells i.e. there may be more
than one copy of the same IS-element in each cell. Altogether IS-elements may account for
about 0.3% of the total bacterial genome. Some of the IS-elements of E. coli that have been
identified and sequenced are IS-1, IS-2, IS-4 and IS-5.

Mechanism of Transposition of IS-Elements:

In transposition of an IS-element or a transposon, the transposable element is precisely

duplicated and one copy is inserted in the target site and the other copy remains at the
original site. Thus, with each transposition event, the number of copies of the element
doubles.

An important feature of transposition of an IS-element is that insertion results in the

duplication of the target site. This site consists of a short DNA segment having 3 to 12
nucleotide pairs. For example, the target site of IS-1 has 9 base pairs and that of IS-2 has 5
base pairs.

The IS-elements are inserted between the two duplicated copies of the target site. Thus, with
each transposition event, the target site is also duplicated. The sequence of bases in the target
site may vary for the same transposable element at different target sites, but the number of
base-pairs remains constant for a particular element.

The process of insertion of an IS-element involves a pair of staggered nicks on the two
opposite sides of the strands of the target DNA producing two free single-stranded ends to
which the copy of the IS-element is joined.

As a result two gaps are created in opposite strands of the recipient DNA. The gaps are filled
up by DNA polymerase and ligase. Thereby, an integrated IS-element with its terminal
indirect repeats is flanked on either side by two short direct repeats originating from the
duplicated target sequence

(4) Phage mu

Bacteriophage Mu is both a virus and a transposon

Phage Mu is a member of the family Myoviridae, with an icosahedral head and contractile
tail.

Its genome, packaged, consists of a single, linear DNA molecule with about 37 000 bp.

In addition, the phage usually packages between 500 and 3000 bp of host DNA, making
phage Mu a generalized transducing phage.

Phage Mu exhibits a temperate life cycle with genome integrated into the chromosome of its
host E. coli.
Bacteriophage Mu, also known as mu phage or mu bacteriophage, is a muvirus (the first
of its kind to be identified) of the family Myoviridae which has been shown to cause
genetic transposition.

Mu phage was first discovered Larry Taylor at UC Berkeley in the late 1950s.

His work continued at Brookhaven National Laboratory, where he first observed

the mutagenic properties of Mu

Several colonies of Hfr E. coli which had been lysogenized with Mu seemed to have a
tendency to develop new nutritional markers

It is after integration that the Mu genome displays an unusual alternate mode of movement,
acting as a transposon that moves from one location in the host genome to another.

It can do this in a nonreplicative manner, leaving one location and inserting into another, or it
can utilize a replicative transfer mode and a second copy of the genome/transposon is then
created at the second location.

Mu-like prophage have been found in other bacteria including Pseudomonas, Haemophilus
influenzae, Neisseria meningitidis, and Deinococcus radiodurans.
Bacteriophage Mu attaches to the E. coli cell and injects its DNA into the cytoplasm. Once
inside, the Mu DNA inserts into the host chromosome via transposition. Notice that the
flanking DNA from the previous host is not inserted. Once integrated, Mu DNA may undergo
so many transpositions that cellular functions are destroyed. When Mu DNA is packaged into
virus particles, short lengths of host chromosome remain attached to the Mu ends; therefore,
Mu DNA is always integrated into host DNA.

Mechanisms

The mechanism of Mu transposition has been deciphered in vitro on both supercoiled and
oligonucleotide substrates.

Figure shows transposition events in the context of in vivo substrates

Mu transposition has two distinct phases, which differ in donor substrate configuration and in
the fate of the transposition products.
During the infection phase, the Mu DNA injected into cells has a peculiar structure. This
DNA is linear in the phage heads, and flanked by non-Mu host DNA acquired during
packaging of integrated Mu replicas during the lytic cycle in a prior host. 60 – 150 bp of host
sequences flank the left or L end of Mu, and 0.5 – 3 kb flank the right or R end.

An injected phage protein N binds to the tip of the flanking DNA (FD), protecting the open
ends from degradation while also converting the linear genome into a non-covalently closed
supercoiled circle prior to integration into the host chromosome.

Non-Mu flanking DNA will be referred to as FD, irrespective of whether the donor substrate
is phage, prophage, plasmid or oligonucleotide.

During the lytic phase, Mu is part of a large covalently closed circular host genome. Thus, the
donor Mu DNA configuration in the infection phase is different from that during the lytic
phase.

In both phases, the mechanism of Mu transposition is the same.

The chemical steps of cleavage and Strand Transfer are the same in both the infection and
lytic phase of transposition

In the infection phase, the linear donor Mu genome is converted to a non-covalently closed
circle, joined by the MuN protein. The E. coli genome is the target.

The Strand Transfer intermediate formed during intermolecular transposition is resolved by

removal of the flanking DNA (FD), and repair of the 5 bp gaps in the target by limited
replication at the host-Mu junction.

In the lytic phase, Mu is part of the covalently closed circular E. coli genome. The ST
intermediate formed during intramolecular transposition is resolved by replication across Mu.