Transcript 1. If the inside ends

微生物遺傳與生物技術
(Microbial Genetics and
Biotechnology)
金門大學
食品科學系
何國傑 教授
Autonomously replicating
genetic entities
(3) the transposable
elements
Transposons
※ Transposons are DNA elements that can hop, or
transpose, from one place in DNA to another. They
are also called “jumping genes”. They carry the
enzyme, transposase responsible for transposition,
the movement by a transposon.
※ They are discovered by Barbara McClintock in the
early 1950s.
※ The transposons now exist in all organisms on the
earth, including human.
※ Transposons may offer a way of introducing genes
from one bacterium into the chromosome of
another bacterium to which it has little DNA
sequence homology, so they obviously play an
important role in evolution.
Transposons
※ Transposition must be tightly regulated and occur
only rarely; otherwise, the cellular DNA would
become riddled with the transposons, which would
have many deleterious effects. In fact, the
frequency of transposition varies from once in
every 103 to once in every 108 cell division,
depending on the type of transposon. It is not
higher than the chance of a gene inactivated by
other mutation. Almost half of human genome may
be the transposons.
※ Genome – The complete DNA sequence
of an organism.
人類基因體計畫
人類的染色體為23對，其一半即為構成人類
的基因體，約含有3 X 109鹼基對（bp），其
大小約為大腸桿茵（4.2 X 106 bp）的1,000
倍，這是否表示人類的基因體所含的基因數
目為大腸桿茵的1,000倍（大腸桿茵有2,000
個基因）。答案是否定的，人類基因體計畫
的完成顯示人類的基大約有35,000個。所以
人類基因體含有大量的〞廢物DNA（junk
DNA）〞，約佔人類總DNA的97％，這些DNA包
括基因中的隱子，基因間的重覆序列及所謂
的跳躍基因。
The largest of component of the human genome
consists of transposons. Other repetitive sequences
include large duplications and simple repeats.
22%
Overview of transposition
I. Types of bacterial tansposons
1. Insertion sequence (IS) elements
(1) These transposons are usually only about 750 ~ 2000 bp
long and encode little more than the transposae that
promote they transposition.
(2) Repeats at ends, usually inverted repeats (shown by arrows).
(3) IS3 consists of two open reading frames (ORFA and ORFB).
(4) ORFB is shifted -1 relative to ORFA, but a programmed -1
(2) Fig.9.2 is the structure of the IS3 element, which contains
frameshift causes the synthesis of a fusion protein, ORFAB,
which is the active transposase.
(5) Smaller protein made from ORFA when the frameshift does
not occur regulates transcription of transposase.
(6) The target site sequence that is duplicated on the insertion
of IS3 is 3 bp long (ex., ATT). The length of such direct
repeats is characteristic of each type of transposon.
Structure of the insertion sequence element IS3
and its related family member
1. The inverted repeats are shown as arrows, and the 3-bp target
sequence that is duplicated after transposition is boxed.
2. OFRA and OFRB encode the N terminus and C terminus of the
transposase, which are translated in different reading frames and are
not active by themselves.
A programmed -1 frameshift put both ORFA
and ORFB in the same frame and makes the
active transposase
The C terminus of the IS3 transposase contains the
DDE motif characteristic of this type of transposase.
I. Types of bacterial tansposons
2. Composite transposons
(1) A larger transposon: Two IS elements of the same
type bracket other genes, usually the antibiotic
resistant gene(s).
(2) Transposition
* Each IS element of the same transposon can
transpose independently as long as the
transposase acts on both of its ends.
* Two IS elements are often not completely
autonomous, because the active transposase of
one IS element can act on the outside ends to
promote transposition of the composite
transposon when the transposase of the other
element is inactive due to a mutation.
I. Types of bacterial tansposons
2. Composite transposons
i. Outside-end transposition
When a transposase acts on the inverted
repeats at the farthest ends of a composite
transposon, the two IS elements transpose
as a unit along with the genes between
them.
ii. Inside-end transposition
A transposase encoded by one IS element can
also act on the inside ends of both IS elements.
Structures of some composite
transposons
1. The active transposase gene is in one of the two IS elements.
2. The IS elements can be in either the same or opposite
orientation (arrows).
Two IS elements can transpose any DNA
between them
Either the outside or inside ends of the IS
elements in a composite transposon can be
used for transposition
(a) Transposition with
the outside ends of
IS10 element would
move Tn10, with the
tetracycline
resistance gene
(Tetr) to another
DNA.
(b) Transposition from the inside ends would create a new composite
transposon carrying the Ampr gene and the plasmid origin of
replication (Ori) to another DNA. (b)If this new composite transposon
hops into a target DNA that does not have a functional origin of
replication, it may confer on that DNA the ability to replicate.
Rearrangements of DNA caused by composite
transposons through inside-ends transposition
The neighboring
sequences between
The original site of
insertion of the
transposon and
the site into which it is
trying to transpose
will be either deleted
or inverted. (B, C, D)
1. If the inside ends (i and ii) do not cross over each other before
they attach (join), the neighboring sequences will be deleted.
2. If the inside ends cross over each other before they attach (join),
the neighboring sequences will be inverted.
3. The DNA between the two IS elements in the composite
transposon will also be deleted.
Rearrangements of DNA caused by composite
transposons through inside-ends transposition
4. Methods have been developed to select tet-sensitive
derivatives of E. coli haerboring the Tn10 transposon. Most of
these tet-sensitive derivatives have deletions or inversions of
DNA next to the site of insertion of Tn10 element.
5. Inside-end transposition is presumably responsible for most of
the often-observed instability of DNA (rearrangement) caused
by composite transposon.
6. Some composite transposons have mechanisms to avoid
inside-end transposition. Ex., adenines of inside-ends of Tn5
are methylated so that they are recognized less well by the
transposase.
7. Assembly of plasmids by IS elements
- Many of the resistance gene on plasmids are bracketed by the
same IS element. Apparently, the plasmid was assembled in
nature by resistance genes hopping onto the plasmid from
some other DNA via the bracketing IS elements.
Rearrangements of DNA caused by composite
transposons through inside-ends transposition
7. Assembly of plasmids by IS elements
- Many of the resistance gene on plasmids are bracketed by the
same IS element. Apparently, the plasmid was assembled in
nature by resistance genes hopping onto the plasmid from
some other DNA via the bracketing IS elements.
III. Mechanisms of transposition
IIIa. A molecular model for transposition of Tn3 (A
replicative transposition)
1. Breaks are made in the target DNA and at the ends of the
transposon, respectively (1 and 2).
2. The 3’ OH ends of the transposon (dots) are ligated to 5’
PO4 ends of the target DNA (3). The insert (3’) shows
details of the ends.
3. The free 3’ ends of the target DNA prime replication in both
directions over the transposon to form the cointegrate (4).
4. The cointegrate is resolved by recombination promoted by
the resolvase TnpR at the res sites (5)
5. Resolution of the cointegrate give rise to two copies of the
transposons, one at the former (or donor) site and a new
one at the target site.
(The A and B in the target DNA illustrate how the target
DNA is reversed in the step 3 for ease of drawing.)
A molecular model for transposition of Tn3
(A replicative transposition)
III. Mechanisms of transposition
IIIa. A molecular model for transposition of Tn3 (A
replicative transposition)
6. The transposase cuts the target and donor DNAs and
promotes ligation of the ends.
7. The resolvase specifically promotes recombination
between trhe res elements in cointegrate.
8. Mu phage replicate itself and insert itself around the
chromosome of its bacterial host by a mechanism similar
to Tn3.
(1) It does not resolve the cointegrate and soon the
chromosome becomes riddled with Mu genome.
(2) These genomes are packaged directly from
chromosomal DNA into the phage head, discarding the
bacterial chromosomal DNA between the inserted Mu
genomes.
IIIb. Transposition by Tn10 and Tn5
• Transposition by a cut-and-paste mechanism
(also known as conservative mechanism or
nonreplicative transposition)
• The transposon is moved from one place and
inserted into another place.
• Transposon produces a short duplication of
target DNA at the ends of the transposon.
• Donor DNA probably leaves break and
is consequently degraded.
• There is no cointegrate formation as it does in
the replicative mechanism.
Cut-and-paste transposition
Details of transposition by DDE
transposons
• All of transposns discussed so far are
considered DDE transposons, because their
trasposaes all have two aspartates (D) and one
glutamate (E) that are essential for their activity.
• These acidic amino acids are not next to each
other in polypeptide, but they are together in the
active center when the protein is folded.
• They hold two Mg2+ that participate in the
cleavage of DNA during the transposition event.
Details of the mechanism of
transposition by Tn5
1. Single copies of the transposae (TnP) bind to each end
of the transposon, and then bind to each other, bring the
two ends of transposon together (synapsis).
2. Transposase bound at one end cuts the DNA at the
other end and vice versa to leave 3’ OH ends at each
end of transposon.
3. These activated 3’ OH ends attack the phosphodiester
bond on the other strand, forming 3’-5’ phosphodiester
hairpins. This cuts the transposon out of the donor DNA.
Details of the mechanism of
transposition by Tn5
4. When the transposase binds to the target DNA, it cuts
the two hairpin ends again and the 3’ OH ends attack
phosphodiester bonds 9 bp apart in the target DNA,
cutting them, and the 5’ phosphate ends in the target
DNA are joined to the 3’ OH ends in the transposon,
inserting the transposon into the target DNA.
5 .The 9-bp single-stranded gaps on each side of
transposon are filled in by DNA polypomerase to make
the 9-bp repeats in the target DNA.
Details of the mechanism of
transposition by Tn5
Details of the mechanism of
transposition by Tn7
• The cut and paste transposon Tn7 can be converted into a
replicative transposon by a single amino acid change in one
subunit of transposase.
• Different subunits of transposase make the cuts in the opposite
strands of DNA at the ends of transposon.
- TnsA cuts at the 5’ and, and TNsB cuts at 3’ end,
- They cut the donor DNA only in the presence of the target
DNA.
• If the TnsA subunit that makes the cut that leaves the 5’
hydroxyl end is altered by a mutation, transposase will cut only
the other strand, leaving a free 3’ OH like a replicative
transposase.
• Apparently, the transposases need only make the appropriate
cuts and joinings, and the replication apparatus of the cell does
the rest.
Details of the mechanism of
transposition by Tn7
• The linear Tn7
transposon
does not cut
itself out of the
donor DNA
unless the
target DNA is
already bound
to the
transposase, so
the 5’ ends are
not left exposed
for long.
The difference and similarity between
replicative and nonreplicative transposition
• The major difference between replicative and nonreplicative
transposition
1. Replicative transposase cuts only one strand at the junction.
2. Nonreplicative transposase makes cuts in both strands in
the junction.
• The similarity between replicative and nonreplicative
transposition
1. The cut 5’ ends of the target DNA are joined to the free 3’
ends of the transposon.
2. The free 3’ ends of target DNA are used as primers for
replication that proceeds until a free 5’ end in the donor DNA
is reached (The only different is whether the replication has
to proceed over the entire transposon (replicative ) or only
over the short region that is duplicated (cut and paste).
IV. General properties of transposons
• Target specificity
1 . No transposable element inserts completely randomly into
target DNA: Target specificity of some transposons are
relaxed and some are stringent.
2. Tn7 transposes with a high frequency into only one site in E.
coli DNA, called attTn7, just downstream of the glmS gene.
(1) The transposition machinery consists of five proteins:
i. TnsA and TnsB – make up the transposase that cuts and
joins the DNA strands.
ii. Other proteins play ancillary roles:
(i) TnsD – may direct the Tn7 to the target sequence,
attTn7. It may induce changes representative of
triple-stranded structures in the attTn7 site.
(ii) TnsC – (i) event directs TnsC to stimulate
transposition into the site.
IV. General properties of transposons
(iii) TnsE – In the absence of TnsD, TnsE
stimulates transposition into other site on
chromosome. This transposition is
inefficient but random.
(2) The glmS gene is highly conserved.
i. The product of glmS performs an important
step in cell wall biosynthesis.
ii. The insertion site of Tn7 is downstream the
gene, and has no effect on cell only its
transcription termination site.
• Effects on genes adjacent to the insertion site –
could be negative or positive (Tn5 and Tn10 contain
promoter near their termini)
IV. General properties of transposons
• Regulation of transposition – transposition of most
transposons occurs rarely because they self- regulate their
transposition.
The regulatory mechanisms differ greatly:
1. Tn3 – The TnpR protein represses the transcription of the
transposase gene (Tnp).
2. Tn10 – transposition occurs primarily just after a
replication fork has passed through the element.
(1) Newly replicated E. coli DNA is hemimethylated at
GATC sites, and it not only activates the transposase
promoter but also increases the activity of the
transposon ends.
(2) The translation of transposase is also repressed by an
antisense RNA.
3. Tn5 - Using a truncated transposase version to inhibit the
active one.
Regulation of transposition
• Regulation of Tn5 transposition
1. Two similar IS50 elements flank the antibiotic resistance genes.
2. An N terminally truncated Tnp (transposase) inhibit the active
one.
3. Dam methylation of the inside ends (IEs) of the IS50 prevents
the transposase from cutting IEs and transposing the
individual IS50 elements.
• OE, outer ends; Inh, inhibitor of transposase Tnp; The dashed
lines indicate that Tnp and Inh made from IS50L are defective.
IV. General properties of transposons
•
Target immunity – Some transposons prefer not to hop close in the
DNA to another transposon of the same type. Immunity can extend
over 100,000 bp of DNA.
1. If two transposons were to insert close to each other, would
cause large deletions and often lead to the death of the cell.
Also, the presence of two transposons close to each other
can cause instability in chromosome.
2. Only Mu, Tn3 and Tn7 families of transposons are known to
exhibit target immunity.
(1) MuB protein seems to be indirectly resposible for the immunity.
(2) The binding of MuB to a DNA make it a target for the MuA
transposase, which then promotes transposon into DNA.
(3) The binding of MuA then cause MuB to dissociate from DNA.
(4) Once a transposon has inserted, a copy of MuA may remain
bound to the end of the inserted transposon, and prevent the
binding of other MUB to the same target DNA and other
transposition into that DNA.
(5) A similar mechanism may explain target site immunity by Tn7,
and the resposible proteins are TnsB and TnsC.
IV. General properties of transposons
• Target immunity – Some transposons prefer
not to hop close in the DNA to another
transposon of the same type.
• Immunity can extend over 100,000 bp of DNA.
1. If two transposons were to insert close to
each other, would cause large deletions
and often lead to the death of the cell.
2. Also, the presence of two transposons
close to each other can cause instability
in chromosome.
V.Transposon mutagenesis
• Transposons are useful for mutagenesis should
have the following properties:
1. It should transpose at a fairly high frequency.
2. It should not be very selective in its target
sequence.
3. It should carry an easily selectable gene, such as
one for resistance to an antibiotic.
4. It should have a broad host range for transposition
if it is to be used in several different kinds of
bacteria.
Transposon Tn5 mutagenesis
Transposon Tn5 mutagenesis
• Transposon Tn5, for example in many types of G – bacteria.
• There are no equally universal methods for G + bacteris.
(A) A standard protocol for transposon mutagenesis of
G- bacteria:
1. A suicide ColE1-derived plasmid contains a mob site and
transposon Tn5 .
2. The relaxase of this suicide plasmid recognizes the coupling
protein of promiscuous plasmid RP4.
3. This suicide plasmid is mobilized into the bacterium by the
products of the RP4 transfer genes, which are inserted in the
chromosome.
4. Tn5 hops into the chromosome of the recipient cell, and the
ColE1 plasmid is lost because it can not replicate.
Transposon Tn5 mutagenesis
(B) Random transposon mutagenesis of a plasmid
1. Transposon Tn5 is introduced into cells on a suicide vector.
2a. The culture is incubated, allowing the Tn5 time to hop, either
into the chromosome (large circle) or into a plasmid (small
circle).
2b. Plating on kanamycin-containing medium results in the
selection of cells in which a transposition has occurred.
3. Plasmid is prepared from Kanr cells and used to transform
Kans cells.
4. Selection for Kanr allows the identification of cells that has
acquired a Tn5-carrying plasmid.
Cloning genes mutated with a transposn
insertion
• A transposon used for mutagenesis of a
chromosome contains a plasmid origin of
replication (ori).
• The chromosome is cut with a restriction
enzyme, ex., EcoRI which no cut in transposon,
and religated.
• Transform E. coli with ligated mix, the resulting
plasmid in the Ampr transformants will contain
the chromosomal sequences that flanked the
transposon .
Cloning genes mutated with a transposn
insertion