Mobile DNA elements

Mobile DNA elements
Transposable elements in bacteria. IS elements. Composite transposons.
Bacteriophage Mu transposition. Tn 10 transposition. SINES and LINES.
Retroviruses and retroposons.

Contents :
1. Introduction.
2.Discovery of Mobile DNA Elements.
3. Types of Mobile DNA Elements.
4. Transposable Elements in Prokaryotes –
a. IS Elements
b. Composite transposon
c. Bacteriophage Mu
d. Tn 10
5. Transposable Elements in Eukaryotes –
a. Retrovirus.
b. LTR Retrotransposon
c. Non LTR Retrotransposon
6. Summary.
7. References.

Introduction.
 The discovery of mobile genetic elements came as a big surprise for scientists. Till this discovery, the genetic information contained in an organism or a cell was
considered to be stable unless mutagenic events caused a change. However, mobile genetic elements do not seem to require any outside interference they move
of their own accord. This means that our genomes are not as static as they seem.
 The other name for mobile genetic elements in transposons. By definition, transposons are those genetic elements which can move around to different positions
the genome of a single cell.
 These elements excise themselves from their original place and then integrate themselves within the same genome but in a different position. In the process they
can cause mutations,
 All transposons have inverted repeats at their ends. But how do these transposons move from one place to another? The phenomenon of transposition requires an
enzyme which helps the mobile element excise itself and in reintegration into another position.
 This enzyme is called transposase and is encoded in a gene that normally is found in the middle of a transposon. If a given transposon does not contain a gene
for transposase, it can use the gene of another transposon and utilize the enzyme produced by this gene for its own mobility.

Transposons
 Transposons are found in both prokaryotes and eukaryotes.
 The bacteria possess four kinds of transposons: insertion sequences, transposons, composite transposons, and bacteriophage elements.
 The eukaryotic transposons are broadly similar to the prokaryotic ones. However, there is an additional group here which is given the name retroposons or
retrotransposons.
 Retroposons are like retroviruses in eukaryotic cells, but are not infectious.
 Except for insertion sequences, other transposons contain genes that are host genes or have some cellular function affecting the host.
 All transposons have (1) inverted repeats at their ends, and (2) genes for transposition in the middle.
 The inverted repeats are about 15-25 bp long, and can be separated by hundreds of base pairs.
 The genes (two or more) in the middle are required for transposition. They encode for proteins called transposase.
 Transposons also have direct repeats at their ends where they have insertede themselves into the host genome.
 Transposons can transpose in one of two ways:
 Conservative transposition, where the entire double-stranded DNA segment moves to a different site in the genome.
 Replicative transposition, where the transposon is first replicated and the one copy stays while the other relocates.
 Transposons are sometimes called "selfish DNA" because they replicate at expense of the host cell (particularly insertion sequences, who do not benefitcell at
all).
 Transposons can cause dna to mutate by insertions, deletions, inversions, breaks in the chromosome, translocation, etc
 Transposon insertion is basically independent of DNA sequence - it is therefore termed as illegitimate recombination.

DISCOVERY OF MOBILE DNA ELEMENTS
 Barbara Mcclintock discovered the first mobile elements while doing classical genetic experiments in maize (corn) during the 1940s.
 Barbara Mcclintock conducted experiments on corn (zea mays) in the united states in the mid-twentieth century to study the structure and function of the
chromosomes in the cells.
 Mcclintock’s original discovery of mobile elements came from observation of certain spontaneous mutations that affect production of any of the several
enzymes required to make anthocyanin, a purple pigment. Mutant kernels are white, and wild-type kernels are purple.
 She characterized genetic entities that could move into and back out of genes, changing the phenotype.
 Her theories were very controversial until similar mobile elements were discovered in bacteria.

TYPES OF MOBILE DNA ELEMENTS
1. DNA TRANSPOSONS –
* Transpose directly as DNA .
* DNA transposons transpose by a “cut-and-paste” mechanism.
* DNA transposons excise themselves from one place in the genome, leaving that site and moving to another.
2. RETROTRANSPOSON –
* Transpose via an RNA intermediate transcribed from the mobile element by an RNA polymerase and then converted back
into double-stranded DNA by a reverse transcriptase
* Retrotransposons make an RNA copy of themselves and introduce this new copy into another site in the genome, while
also remaining at their original position.
* Retrotransposons move by a “copy-and-paste” mechanism in which the copy is an RNA intermediate.
* The movement of retrotransposons is analogous to the infectious process of retroviruses

Two major classes of mobile
elements.
(a) Eukaryotic DNA transposons
(orange) move via a DNA
intermediate, which
is excised from the donor site.
(b) Retrotransposons (green) are first
transcribed into an RNA
molecule, which is then reverse-
transcribed
into double-stranded DNA. In both
cases, the
double-stranded DNA intermediate is
integrated
into the target-site DNA to complete
movement.
Thus DNA transposons move by a
cut-and-paste
mechanism, whereas
retrotransposons move by
a copy-and-paste mechanism.

TRANSPOSABLE ELEMENTS IN PROKARYOTES.
* There are 3 types of transposable elements in prokaryotes –
1 Insertion sequence.
2 Transposons.
3 Bacteriophage Mu
TRANSPOSABLE ELEMENTS IN BACTERIA.
* There are 3 main types of bacterial transposons –
1 Insertion sequence or IS Elements.
2 Composite Transposons.
3 Tn3 like Elements.

* The IS Elements are the simplest, containing only genes that encode proteins involved in transposition.
* The composite transposons and Tn3 like elements are more complex , containing some genes that encode products unrelated to transposition
process.
IS ELEMENTS IN BACTERIA
* The first molecular understanding of mobile elements came from the study of certain E. coli mutations caused by the spontaneous insertion of
a DNA sequence, about 1–2 kb long, into the middle of a gene. These inserted stretches of DNA are called insertion sequences, or IS elements.
* These are transposable sequences which can insert at different sites in bacterial chromosomes.
* IS Elements contains ITRs [ Inverted terminal repeats ].
* IS Elements are relatively short usually not exceeding 2500 bp.
* The ITRs present at the ends of IS-elements are important feature which enables their mobility.
* The ITRs present in the IS elements of E.coli usually range between 18-40 bp.

* The term Inverted Terminal Repeat [ITR]implies that the sequence at 5 end of one strand is identical to the sequence at 5`end of the other
strand but they run in inverse opposite direction.

* General structure of bacterial IS elements.
* The relatively large central region of an IS element, which encodes one or two enzymes required for transposition, is flanked by an inverted
repeat at each end.
* The sequences of the inverted repeats are nearly identical, but they are oriented in opposite directions. The sequence is characteristic of a
particular IS element.
* The 5′ and 3′ short direct repeats are not transposed with the insertion element; rather, they are insertion-site sequences that become
duplicated, with one copy at each end, during insertion of a mobile element.
* The length of the direct repeats is constant for a given IS element, but their sequence depends on the site of insertion and therefore varies with
each transposition of the IS element. Arrows indicate sequence orientation.

* Between the inverted repeats is a protein-coding region, which encodes one or two enzymes required for transposition of an IS element to a
new site.
* An important hallmark of IS elements is the presence of short direct repeats, containing 5 – 11 base pairs, immediately adjacent to both ends of
the inserted element.
* The length of the direct repeat is characteristic of each type of IS element, but its sequence depends on the target site where a particular copy of
the IS element is inserted.
* Duplication of this target-site sequence to create the second direct repeat adjacent to an IS element occurs during the insertion process.
* Transposition of an IS element occurs by a “cut-and-paste” mechanism.
* Transposase performs three functions in this process:
* (1) It precisely excises the IS element from the donor DNA,
* (2) Makes staggered cuts in a short sequence in the target DNA, and
* (3) ligates the 3′ termini of the IS element to the 5′ ends of the cut target DNA. Finally, a host-cell DNA polymerase fills in the single-stranded
gaps, generating the short direct repeats that flank IS elements, and DNA ligase joins the free ends.*

Step 1 : Transposase, which is encoded by the IS
element (IS10 in this example), cleaves both strands of
the donor DNA next to the inverted repeats (dark red),
excising the IS10 element. At a largely random target
site, transposase makes staggered cuts in the target
DNA. In the case of IS10, the two cuts are 9 bp apart.
Step 2 : Ligation of the 3′ ends of the excised IS
element to the staggered sites in the target DNA is also
catalyzed by transposase.
Step 3 : The 9-bp gaps of single-stranded DNA left in
the resulting intermediate are filled in by a cellular DNA
polymerase;
finally, cellular DNA ligase forms the 3′→5′
phosphodiester bonds between the 3′ ends of the
extended target DNA strands and the 5′ ends of the IS10
strands.
This process results in duplication of the target-site
sequence on each side of the inserted IS element.

Bacterial composite transposon
* In addition to. IS elements, bacteria contain composite mobile genetic elements that are larger than IS elements known as composite transposons
and are shown by the symbol `Tn`.
* It is made up of 2 IS Elements , one present at each end of a DNA sequence , which contains genes whose function s are not related to the
transposition process.
*contain one or more protein-coding genes in addition to those required for transposition.
*these elements are composed of an antibiotic-resistance gene flanked by two copies of the same type of IS element.
*Insertion of a transposon into plasmid or chromosomal DNA is readily detectable because of the acquired resistance to an antibiotic.
* These transposons have ITRs at the ends.
*Transposition produces a short direct repeat of the target site on either side of the newly integrated transposon, just as for IS elements.
*It can be said that these are the large transposons which are formed by capturing of an immobile DNA sequence within 2 insertion sequence thus
enabling it to move.
*Example of such transposons include the members of Tn series like Tn1, Tn5, Tn9, Tn10 etc.

* General structure of bacterial transposons, such as Tn9 of E. coli.
* 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 .
* 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

Tn 10 transposon
* Tn10 is a composite transposon.
* It comprises a pair of IS10 insertion sequences located in opposite orientation flanking ~6.7 kb of unique sequences; these unique sequences
encode a tetracycline resistance determinant and other determinants whose functions remain to be identified .

* One of Tn10’s two IS10 elements, IS10-Right, is structurally and functionally intact and is considered to be the “wild type” IS10.
* IS10 encodes a single transposase protein which mediates transposition by interacting with specific sequences at two oppositely oriented IS10
(or Tn10) termini.
* The termini of IS10 are subtly different and are referred to as the “outside” and “inside” end, respectively, by virtue of their position in Tn10.
* IS10-Left is structurally intact but encodes a substantially defective transposase.
* Tn10 has a composite structure and it is composed of a pair of insertion sequence elements (IS10) flanking five genes.
* Only one of the IS10 elements encodes a functional transposase.[Since the ends of the IS10 element contain the transposase recognition sites,
Tn10 has a total of four such sites.
* If the transposase binds the two recognition sites flanking an IS10 element, the IS10 element undergoes transposition independently of the larger
composite structure.
* If the transposase binds the two outermost recognition sites, the whole composite Tn10 structure undergoes transposition.

Two of the five genes encoded by the central portion of Tn10, tetA and tetR confer resistance to the antibiotic tetracycline.
The functions of the other three genes, jemA, jemB and jemC, are unknown .
The Tn10/IS10 transposase is closely related to another composite transposon, Tn5/IS50.
Transposition in Tn 10
The composite bacterial transposon Tn10 transposes by a non-replicative mechanism.
The complete transposon, or one of its two flanking IS10 modules, is first excised by a pair of double strand breaks and then inserted into a new
target site.
These steps are carried out by the Tn10-encoded transposase protein, a 46 kDa protein that binds to specialized DNA sequences located at
transposon termini or ‘ends’

Non-replicative, "cut-and-paste" mechanism
.* 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. A cartoon of this process is shown in
previous slide.
* Non-replicative transposition of Tn10 involves three distinct chemical steps,
* 1 first-strand nicking
* 2 hairpin formation
* 3 hairpin resolution.
* Tn10 transposition involves the formation of a hairpin intermediate at the transposon termini.

Structure of IS10 Right and transposase-mediated
chemical steps in Tn10/IS10 transposition.
IS10 Right (thick lines) encodes transposase protein
and contains the binding determinants for
transposase (filled rectangles) at its termini, the
‘inside’ (IE) and ‘outside’ (OE) end sequences.
The inverted repeat sequence (half arrows) contains
the primary specificity determinants for transposase
binding. In addition, the OE also contains a binding
site for Escherichia coli IHF (open rectangle) and a
second ‘distal’ transposase-binding site.
The four chemical steps that occur at each
transposon end are indicated; thin lines indicate
flanking donor DNA.
The top strand shown is the ‘non-transferred’ strand
(NTS) and the bottom strand is the ‘transferred’
strand (TS).

First, hydrolytic cleavage of the transferred strand at the terminal nucleotide would generate a 3′OH group.
Second, this 3′OH would attack the non-transferred strand, joining the terminal 3′OH to the scissile phosphate to form a hairpin at the
transposon end and releasing the flanking donor DNA as a double-stranded end.
Third, the hairpin would be resolved by hydrolytic attack at the terminal phosphodiester linkage to regenerate the 3′OH residue at nucleotide
1 of the transferred strand. These steps would be followed by strand transfer.

BACTERIOPHAGE Mu
 Bacteriophage is a type of virus that infect
bacteria, also known as phage Mu.
 Since it infects the members of enterobacteria it is called enterobacteria
phage Mu.
 It belongs to family Myoviridae and it is dsDNA virus.
 It is a temperate and transposable phage which causes transposition of genes
at the time of its multiplication cycle.
 Bacteriophage Mu is both a virus and a transposon.
 When Mu DNA enters E. coli, its bacterial host, it integrates at random
sites into the host chromosome by transposition.
 In other words, the whole of the Mu genome is a transposon.

If Mu inserts into the middle of a host gene this will be inactivated. Early investigators noticed that infection with this virus caused frequent mutations
and therefore named it Mu for “mutator” phage.
* It is the longest transposon known so far.
* Caries numerous gene for viral head and tail formation.
* Replication of Mu produces about 100 viral chromosomes in a cell , arises from transposition of Mu to about 100 different target sites.
* Therefore considered as giant mutator transposon.
* It has an icosahedral head and contractile tail.
* Its packaged genome consists of a single, linear dsDNA molecule with about 37,000 base pairs.
* In addition, the phage usually packages between 500 and 3000 base pairs of host DNA from random chromosomal locations, making phage Mu a
generalized transducing phage.
* Phage Mu exhibits a temperate life cycle with its genome integrated into the chromosome of its host E. coli.

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 non-replicative 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 created at the second location.
* Mu integrates into its E. coli host by transposition. Mu may exist as a prophage or enter into the lytic cycle.
* Mu undergoes replicative transposition when it replicates and produces many copies of itself, that ultimately destroy the host.
* However, Mu does have a mechanism in place to ensure that it does not integrate into its own genome and destroy itself. This is called
transposition immunity and relies on the binding of specific Mu proteins.
* Mu is a lysogenic phage, capable of inserting into the host chromosome like a transposon.
* In the lytic cycle, Mu replicates by transposing randomly across the host chromosome. The transposition of Mu during the lytic cycle is
estimated to occur about 100 times, revealing a robust transposition mechanism.

Mu Transposition Mechanism
Mu transposition has two distinct phases – 1. Infection phage
2. Lytic phage
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 .
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.

A) In the infection phase, the linear donor
Mu genome is converted to a non-
covalently closed circle, joined by the
MuN protein (purple ovals; ends shown
unjoined for clarity); the E. coli genome
is the target. The ST 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.
(B) 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.

* The transposase MuA initially generates a pair of water-mediated endo-nucleolytic cleavages on specific Mu-host phosphodiester bonds,
producing 3’-OH nicks at Mu DNA ends .
* In the subsequent strand transfer (ST) step, the 3’-OH ends directly attack phosphodiester bonds in the target DNA spaced 5 bp apart; this reaction
is intermolecular in the infection phase and intramolecular in the lytic phase .
* Mu ends join to 5’-Ps in the target, leaving 3’-OH nicks on the target. The MuB protein is essential for efficient capture of the target, but plays
critical roles at all stages of transposition by allosterically activating MuA .
* The cleavage and ST reactions, also called phosphoryl transfer reactions are common to other DNA transposition systems including retroviral
integration .
* These reactions take place within the same active site of MuA, which contains a structurally conserved ‘DDE’ domain.
* Post-transposition, the branched ST intermediate product must be resolved .
* During the infection phase, the intermediate is resolved by FD removal/degradation and repair of the 5 bp gaps in the target by limited DNA
replication, generating a simple insertion of Mu in the E. coli genome.
* During the lytic phase, the intermediate is resolved by target-primed replication across the entire Mu genome. These product resolution pathways
will be referred to as repair or replication pathways.

TRANSPOSOSOME ASSEMBLY, ACTIVITY, AND STRUCTURE
The Mu transpososome is assembled by interactions of transposase MuA subunits with the left and right ends of Mu and an enhancer located in
between.
* Mu transposition requires MuA binding to sites at the left (L) and right (R) ends (also referred to as att ends), as well as at an enhancer (E) DNA
segment located ~1 kb away from the L end on the Mu genome .
* The L and R ends are asymmetric with respect to orientation and spacing of the three MuA-binding sites at each end (L1-L3 and R1-R3). There are
three MuA-binding sites at E as well (O1-O3).
*Separate regions within MuA bind to the end and enhancer sites .
* Under physiological reaction conditions, transpososome assembly requires that both sets of DNA sites in their native configuration be present on
supercoiled Mu donor DNA, along with the E. coli protein HU, which binds between L1 and L2 at the L end , the E. coli IHF protein, which binds
between O1 and O2 at E, optimizes assembly. In the presence of the divalent metal ion Mg2+, these components are sufficient for promoting the DNA
cleavage reaction.
* ST requires MuB in addition , MuB is an AAA+ ATPase, which binds DNA non-specifically .
* The ATPase activity of MuB is stimulated by DNA and MuA. MuB not only captures target DNA and delivers it to the transpososome, but its
interactions with MuA optimize all stages of transpsosome assembly.

TRANSPOSABLE ELEMENTS IN EUKARYOTES.
* Most mobile elements in eukaryotes are retrotransposons, but eukaryotic DNA transposons also occur.
* Indeed, the original mobile elements discovered by Barbara McClintock are DNA transposons.
* on the basis of mechanism of transposition , mobile genetic elements of eukaryotes can be divided into 2 classes:
* Class I elements- RNA Transposable elements [ DNA to RNA to DNA]
Includes 3 families: 1 Retroviruses.
2 Long terminal repeat [ LTR ] Retrotransposons , Viral family.
3 Non LTR Retrotransposons, Non viral family
* Class II Elements DNA Transposable elements [ DNA to DNA ]

Retroviruses
* The genomes of a number of animal viruses can integrate into the host-cell genome. Among the most important of these are the
retroviruses.
* retroviruses are enveloped viruses with a genome consisting of two identical strands of RNA.
* These viruses are so named because their RNA genome acts as a template for the formation of a DNA molecule—a flow of genetic
information that is opposite to the more common transcription of DNA into RNA.

RETROVIRUS GENOMES Are composed of single stranded RNA comprising at least 3 genes –
* gag – coding for structural proteins of viral particle
* pol – coding for a reverse transcriptase / integrase protein
* env – coding for a protein embedded in the virus`s lipid envelop
* Retrovirus are distinguished from other types of retroelments by the presence of an env gene in their genome .

The protein encoded by this gene allows retroviruses to enter and leave their host cell.
Retroviruses are therefore the only infectious type of retroelement , they spread from cell to cell, and also organism to organism.
pol gene also has a DNA polymerase activity, which enables it to synthesize a duplex DNA from the single stranded RNA . The enzyme has an
Rnase H activity , which can degrade RNA part of RNA-DNA hybrid.
. In the retroviral life cycle , a viral enzyme called reverse transcriptase initially copies the viral RNA genome into single-stranded DNA that is
complementary to the viral RNA; the same enzyme then catalyzes the synthesis of a complementary DNA strand.
The resulting double-stranded DNA is integrated into the chromosomal DNA of the infected cell by an integrase enzyme in the virion.
Finally, the integrated DNA, called a provirus, is transcribed by the host cell’s own RNA polymerase into RNA, which is either translated into
viral proteins or packaged within virion capsid proteins to form progeny virions that are released by budding from the host-cell membrane.
Because most retroviruses do not kill their host cells, infected cells can replicate, producing daughter cells with integrated proviral DNA. These
daughter cells continue to transcribe the proviral DNA and bud progeny virions.

Retroviral life cycle.
Retroviruses have a genome of two identical copies of single-stranded RNA and an outer envelope.
Step 1 : After viral glycoproteins in the retroviral envelope interact with a specific host-cell membrane protein, the envelope fuses directly with the
plasma membrane, allowing entry of the nucleocapsid into the cytoplasm of the cell.
Step 2 : Viral reverse transcriptase and other proteins copy the viral ssRNA genome into a double-stranded DNA.
Step 3 : The viral dsDNA is transported into the nucleus and integrates into one of many possible sites in the host-cell chromosomal DNA. For
simplicity, only one host-cell chromosome is depicted.
Step 4 : The integrated viral DNA (provirus) is transcribed by the host-cell RNA polymerase, generating viral mRNAs (dark red) and viral genomic
RNA molecules (bright red). The host-cell machinery translates the viral mRNAs into glycoproteins and nucleocapsid proteins.
Step 5 : Progeny virions then assemble and are released by budding.
* Some retroviruses contain cancer-causing genes (oncogenes), and cells infected by such retroviruses are oncogenetically transformed into tumor
cells.
* Among the known human retroviruses are human T-cell lymphotrophic virus (HTLV), which causes a form of leukemia, and human
immunodeficiency virus (HIV-1), which causes acquired immune deficiency syndrome (AIDS).

RETROTRANSPOSONS
* All eukaryotes studied from yeast to humans contain retrotransposons, mobile DNA elements that transpose through an RNA intermediate
utilizing a reverse transcriptase .
* These mobile elements are divided into two major categories, viral and nonviral retrotransposons.
* Viral retrotransposons are abundant in yeast (e.g., Ty elements) and in Drosophila (e.g., copia elements).
* In mammals, nonviral retrotransposons are the most common type of mobile element; Still, viral retrotransposons are estimated to account for
≈4 percent of human DNA.

Viral retrotransposons
General structure of eukaryotic viral retrotransposons
* The central protein-coding region is flanked by two long terminal repeats (LTRs), which are element-specific direct repeats.
* LTRs, the hallmark of these mobile elements, also are present in retroviral DNA.
* Like other mobile elements, integrated retrotransposons have short target-site direct repeats at their 3′ and 5′ ends.

* In addition to the short 5′ and 3′ direct repeats that are typical of all transposons, these retrotransposons are marked by the presence of LTRs
flanking the central protein-coding region.
* These long direct terminal repeats, containing 250–600 bp depending on the particular LTR retrotransposon, are characteristic of integrated
retroviral DNA and are critical to the life cycle of retroviruses.
* In addition to sharing LTRs with retroviruses, LTR retrotransposons encode all the proteins of the most common type of retroviruses, except for
the envelope proteins. Lacking these envelope proteins, LTR retrotransposons cannot bud from their host cell and infect other cells; however, they
can transpose to new sites in the DNA of their host cell.
* Because of their clear relationship with retroviruses, LTR retrotransposons are often called retrovirus-like elements.
* A key step in the retroviral life cycle is the formation of retroviral genomic RNA from integrated retroviral DNA .
* We describe this process in some detail here because it serves as a model for the generation of the RNA intermediate during the transposition of
LTR retrotransposons.

Generation of retroviral genomic RNA from integrated retroviral DNA

* the leftward retroviral LTR functions as a promoter that directs host-cell RNA polymerase to initiate transcription at the 5′ nucleotide of the roughly
20-base R sequence that is repeated at each end of the retroviral RNA.
* After the entire downstream retroviral DNA has been transcribed, the RNA sequence corresponding to the rightward LTR directs host-cell RNA-
processing enzymes to cleave the primary transcript and add a poly(A) tail at the 3′ end of the R sequence.
* The resulting retroviral RNA genome, which lacks a complete LTR, exits the nucleus and is packaged into a virion that buds from the host cell.
* After a retrovirus infects a cell, reverse transcription of its RNA genome by the retrovirus-encoded reverse transcriptase yields a double-stranded
DNA containing the cytosol.
* The double-stranded DNA, with an LTR at each end, is then transported into the nucleus in a complex with integrase, another enzyme encoded by
retroviruses.
* Retroviral integrases are closely related to the transposases encoded by DNA transposons and use a similar mechanism to insert the double-stranded
retroviral DNA into the host-cell genome.
* In this process, short direct repeats of the target-site sequence are generated at either end of the inserted viral DNA sequence.

* Although the mechanism of reverse transcription is complex, it is a critical aspect of the retrovirus life cycle.
* The process generates the complete 5′ LTR that functions as a promoter for initiation of transcription precisely at the 5′ nucleotide of the R
sequence, while the complete 3′ LTR functions as a poly(A) site leading to polyadenylation precisely at the 3′ nucleotide of the R sequence.
* Consequently, no nucleotides are lost from an LTR retrotransposon as it undergoes successive rounds of insertion, transcription, reverse
transcription, and reinsertion at a new site.
* As noted above, LTR retrotransposons encode reverse transcriptase and integrase.
* By analogy with retroviruses, these mobile elements move by a “copy-and-paste” mechanism whereby reverse transcriptase converts an RNA
copy of a donor element into DNA, which is inserted into a target site by integrase.

Generation of LTRs during reverse transcription of retroviral genomic RNA

Generation of LTRs during reverse transcription of retroviral genomic RNA
* A complicated series of nine events generates a double-stranded DNA copy of the single-stranded RNA genome of a retrovirus (top).
* The genomic RNA is packaged in the virion with a retrovirus-specific cellular tRNA hybridized to a complementary sequence near its 5′ end
called the primer-binding site (PBS).
* The retroviral RNA has a short direct-repeat terminal sequence (R) at each end. The overall reaction is catalyzed by reverse transcriptase , which
catalyses polymerization of deoxyribonucleotides. Rnase also encoded in the viral RNA and packaged into the virion particle, digests the RNA
strand in a DNA-RNA hybrid.
* The entire process yields a double-stranded DNA molecule that is longer than the template RNA and has a long terminal repeat (LTR) at each end.
* The most common LTR retrotransposons in humans are called ERVs, for endogenous retroviruses.

Nonviral retrotransposons.
The most abundant mobile elements in mammals are retrotransposons that lack LTRs, sometimes called nonviral retrotransposons.
* These form two classes in mammalian genomes:
* 1 long interspersed elements (LINEs)
* 2 short interspersed elements (SINEs).
LINEs-
* In humans, full-length LINEs are about 6 kb long,
* Human DNA contains three major families of LINEs that are similar in their mechanism of transposition but differ in their sequences: L1, L2, and
L3 .
* Only members of the L1 family transpose in the contemporary human genome; apparently there are no remaining functional copies of L2 or L3.
* LINE sequences are present at roughly 900,000 sites in the human genome, accounting for a staggering 21 percent of total human DNA.

General structure of an L1 LINE element, a common type of eukaryotic nonviral retrotransposon
* LINEs are usually flanked by short direct repeats, the hallmark of mobile elements.
* It contain two long open reading frames :
* 1. ORF1, about 1 kb long, encodes an RNA-binding protein.
* 2. ORF2, about 4 kb long, encodes a protein that has a long region of homology with the reverse transcriptase of retroviruses and LTR
retrotransposons, but also exhibits DNA endonuclease activity.
* The length of the flanking direct repeats varies among copies of the element at different sites in the genome. The sequence of the direct repeats
appears to be generated from the target-site sequence during insertion.
* Although L1 elements do not contain LTRs, the A/T-rich region at the right end is thought to function in retro transposition.

* Evidence for the mobility of L1 elements first came from analysis of DNA cloned from humans with certain genetic diseases.
* DNA from these patients was found to carry mutations resulting from insertion of an L1 element into a gene , whereas no such element occurred in
the DNA of either parent.
* Since L1 elements do not contain LTRs, their mechanism of transposition through an RNA intermediate must differ from viral retro transposition
in which the LTRs play a crucial role.
* transcription of L1 is directed by promoter sequences located within the left end of the element. L1 elements also contain an A/T-rich region near
their right end, which generates a stretch of A residues in their RNA transcripts.
* The vast majority of LINEs in the human genome are truncated at their 5′ end, suggesting that reverse transcription was terminated before
completion .
* Because of this shortening, the average size of LINE elements is only about 900 bp, even though the full-length sequence is about 6 kb long

SINES
* The most abundant class of mobile elements in the human genome, SINEs constitute about 13 percent of total human DNA.
* Varying in length from about 100 to 400 base pairs.
* these retrotransposons do not encode protein, but most contain a 3′ AT-rich sequence similar to that in LINEs.
*SINEs are transcribed by the same nuclear RNA polymerase that transcribes genes encoding tRNAs, 5S rRNAs, and other small stable RNAs.
* Most likely, the ORF1 and ORF2 proteins expressed from full-length LINEs mediate reverse transcription and integration of SINEs .
* Consequently, SINEs can be viewed as parasites of the LINE symbionts, competing with LINE RNAs for binding, reverse transcription, and
integration by LINE-encoded ORF1 and ORF2.

* Because many of these repetitive sequences in human DNA were found to contain a recognition site for the restriction enzyme Alu I, they were
collectively called the Alu family.
*Alu sequences containing ≈300 base pairs are present at ≈1 million sites in the human genome, accounting for about 10 percent of the
total genomic DNA.
*Alu sequences are remarkably homologous to 7SL RNA, a small cellular RNA that is part of the signal-recognition particle. This cytoplasmic
ribonucleoprotein particle aids in the secretion of newly formed polypeptides through the membranes of the endoplasmic reticulum
* The initial evidence for the mobility of SINES came from analysis of DNA from a patient with neurofibromatosis, a genetic disorder marked
by the occurrence of multiple neuronal tumors called neurofibromas due to mutation in the NF1 gene.
* Like the retinal tumors that occur in hereditary retinoblastoma , neurofibromas develop only when both NF1 alleles carry a mutation. In one
individual with neurofibromatosis, one NF1 allele contains an inactivating Alu sequence; inactivating somatic mutations in the other NF1 allele
in peripheral neurons lead to the development of neurofibromas

SUMMARY
Most of the moderately repeated DNA sequences interspersed at multiple sites throughout the genomes of higher eukaryotes arose from mobile DNA
elements.
* Mobile DNA elements encode enzymes that can insert their sequence into new sites in genomic DNA.
* Mobile DNA elements that transpose to new sites directly as DNA are called transposons; those that first are transcribed into an RNA copy of the
element, which then is reverse-transcribed into DNA, are called retrotransposons . Both types generally produce short direct repeats at the site of
insertion, which flank the mobile element. The length of the direct repeats depends on the type of mobile element.
* The mobile DNA elements in bacteria — IS elements and bacterial transposons — move via DNA intermediates. Both encode transposase, but the
longer transposons also contain at least one other protein-coding gene, generally including a drug-resistance gene.
* Although transposons, similar in structure to bacterial IS elements, occur in eukaryotes (e.g., Drosophila P element), retrotransposons generally are
much more abundant, especially in higher eukaryotes.
* Viral retrotransposons are flanked by long terminal repeats (LTRs), similar to those in retroviral DNA, and, like retroviruses, encode reverse
transcriptase and integrase. They move in the genome by being transcribed into RNA, which then undergoes reverse transcription and integration into
the host-cell chromosome.
* Nonviral retrotransposons lack LTRs and have an A/T-rich stretch at one end. These mobile elements are thought to move by an unusual nonviral
retro transposition mechanism.

* The most abundant mobile elements in vertebrates are two types of nonviral retrotransposons called LINES and SINES. Both types appear to
have caused mutations associated with human genetic diseases.
* SINES exhibit extensive homology with small cellular RNAs transcribed by RNA polymerase III. The most common SINES in humans
frequently contain a site for the restriction enzyme Alu I and consequently are called Alu sequences. These ≈300-bp sequences are scattered
throughout the human genome, constituting ≈5 percent of the total DNA.
* Although mobile DNA elements appear to serve no beneficial function to an individual organism, they most likely influenced evolution
significantly.

REFERENCES :
LODISH, MOLECULAR CELL BIOLOGY, CHAPTER – 5, PAGE NO. ( FROM – 258 TO 260 ), CHAPTER – 8, PAGE NO. ( FROM 354 TO 365)
https://www.ncbi.nlm.nih.gov/books/NBK21495/
https://en.wikipedia.org/wiki/Mobile_genetic_elements
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486318/
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/enterobacteria-phage-mu
https://sites.cns.utexas.edu/harsheylab/bacteriophage-mu
https://en.wikipedia.org/wiki/Tn10
https://link.springer.com/chapter/10.1007/978-3-642-79795-8_3
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/transposons/non-repl-tpn.html
https://www.sciencedirect.com/science/article/pii/S0092867400817882
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC125497/

Mobile DNA elements

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