Transposase Binding Research Articles

Tn 3 transposition immunity is conferred by the transposase-binding domain in the terminal inverted-repeat sequence of Tn 3

A series of mutant terminal inverted repeats (IRs), having2 bp subtitutions at various sites within the 38-bp IR sequence of the ampicillin-resistance transposon Tn 3, were tested for transposition immunity to Tn 3. Mutations within region 1–10 in the IR did not affect transposition immunity, while mutations within region 13–38 inactivated the immunity function. These two regions corresponded to domain A which was not bound specifically by Tn 3 transposase and to domain B which was bound by the transposase, respectively. This indicates that specific binding of transposase to domain B within the IR sequence is responsible for transposition immunity.

Two domains in the terminal inverted-repeat sequence of transposon Tn3

Tn3 and related transposons have terminal inverted repeats (IR) of about 38 bp that are needed as sites for transposition. We made mini-Tn3 derivatives which had a wild-type IR of Tn3 at one end and either the divergent IR of the Tn3-related transposon, γδ or IS101, or a mutant IR of Tn3 at the other end. We then examined both in vivo transposition (cointegration between transposition donor and target molecules) of these mini-Tn3 elements and in vitro binding of Tn3-encoded transposase to their IRs. None of the elements with an IR of γδ or IS101 mediated cointegration efficiently. This was due to inefficient binding of transposase to these IR. Most mutant IR also interferred with cointegration, even though transposase bound to some mutant IR as efficiently as it did to wild type. This permitted the Tn3 IR sequence to be divided into two domains, named A and B, with respect to transposase binding. Domain B, at positions 13–38, was involved in transposase binding, whereas domain A, at positions 1–10, was not. The A domain may contain the sequence recognized by some other (e.g., host) factor(s) to precede the actual cointegration event.

Drosophila P element transposase recognizes internal P element DNA sequences

Drosophila P transposable elements encode an 87 kd trans-acting protein, transposase, that is required to catalyze P element transposition and excision. We show here that purified transposase is a site-specific DNA binding protein. P element transposase does not interact with the terminal 31 bp inverted repeats but instead interacts specifically with an internal 10 bp consensus sequence present at both the 5′ and 3′ ends of P element DNA. These binding sites lie within sequences known to be important for transposition in vivo. Transposase also displays an unusually high nonspecific affinity for DNA. The transposase binding site at the 5′ end overlaps sequences we show to be essential for transcription from the P element promoter in vitro, which raises the possibility that either transposase or the related 66 kd P element protein may affect P element transcription. From these and other observations, we suggest that the P element transposition reaction probably requires the binding of additional Drosophila protein factors to the terminal DNA sequences.

Mutational analysis of IS10′s outside end.

We present the genetic analysis of a large number of mutations in the outside end of insertion sequence IS10. (i) The terminal inverted repeat sequence is probably the primary site of transposase binding. Mutations in this region fall into phenotypic classes which correspond to their map locations, suggesting that this region may consist of several distinct functional segments. Similarities between the organization of IS10's inverted repeat and those of other transposable elements are discussed. (ii) Base pairs 23-42 include a consensus binding sequence for one of the IS10 transposition host factors, IHF. The phenotypes of mutations in this region suggest that IHF is the major host factor for outside-end transposition activity in vivo and that base pairs throughout this region are important for the IHF interaction. (iii) Mutations in bp 43-61 do not affect outside-end transposition activity but do affect, in expected ways, previously identified determinants involved in expression and regulation of transposase. (iv) Some mutations in bp 23-42 also affect transposase expression; the possibility that IHF negatively regulates transcription initiation is discussed.

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.

Outreading promoters are located at both ends of the Gamma-delta transposon

Two plasmids were isolated containing oppositely oriented gamma-delta insertions between the wild-type transcription initiation site of the nifHDKY operon and the nifH coding sequence. The nifHDKY promoter of Klebsiella pneumoniae, similar to other nitrogen fixation (nif) promoters, normally requires the products of ntrA and nifA for activity. Mutations that allowed constitutive expression of the nifHDKY operon were searched for by transforming a plasmid, containing the regulatory region of this operon followed by an in-frame nifH'-'lacZ fusion, into a Lac- Escherichia coli strain (which contains no nifA) and screening for Lac+ derivatives. The plasmids described here were isolated from such derivatives and directed the constitutive expression of beta-galactosidase. Deletion analysis indicated that gamma-delta promoters other than those transcribing tnpA and tnpR were involved in this expression. Nuclease S1 mapping revealed outward-reading transcription initiation sites in both the gamma end and the delta end of the transposon. Most interestingly, one initiation site on each end was located in corresponding positions within the terminal inverted repeats. The sites were in the center of the longest sequence, of 12 bp, contiguously conserved between the terminal inverted repeats of gamma-delta and the related transposon Tn3. In gamma-delta and Tn3, this sequence has been recently implicated in transposase binding. These results suggest a possible interrelationship between transcription from the "end" promoters and transposition.

Gamma delta transposase and integration host factor bind cooperatively at both ends of gamma delta.

gamma delta, a prokaryotic transposon, encodes a transposase that is essential for its transposition. We show here, by DNase I protection experiments, that purified gamma delta transposase binds at the transposon's inverted repeats (IRs). Immediately adjacent to each transposase binding site (and within gamma delta DNA) we have identified a binding site for an additional protein factor, the Escherichia coli-encoded integration host factor (IHF). The binding of transposase and IHF to these adjacent sites is mutually cooperative. An IHF binding-site was also found in the original target DNA, just outside one of the ends of gamma delta. The affinity of IHF for this flanking site is reduced by transposase. These results demonstrate that gamma delta transposase binds at the IRs of gamma delta, and suggest that IHF may be involved in forming a transposase-DNA complex and/or influencing the target site selection during the transposition of gamma delta.

Binding of the Tn3 transposase to the inverted repeats of Tn3

The transposase protein and the inverted repeat sequences of Tn3 are both essential for Tn3 cointegrate formation and transposition. We have developed two assays to detect site-specific binding of transposase to the inverted repeats: (1) a nitrocellulose filter binding assay in which transposase preferentially retains DNA fragments containing inverted repeat sequences, and (2) a DNase 1 protection assay in which transposase prevents digestion of the inverted repeats by DNase 1. Both assays show that transposase binds directly to linear, duplex DNA containing the inverted repeats. The right inverted repeat of Tn3 binds slightly more strongly than the left one. Site-specific binding requires magnesium but does not require a high energy cofactor.

Specific binding of transposase to terminal inverted repeats of transposable element Tn3.

Tn3 transposase, which is required for transposition of Tn3, has been purified by a low-ionic-strength-precipitation method. Using a nitrocellulose filter binding assay, we have shown that transposase binds to any restriction fragment. However, binding of the transposase to specific fragments containing the terminal inverted repeat sequences of Tn3 can be demonstrated by treatment of transposase-DNA complexes with heparin, which effectively removes the transposase bound to the other nonspecific fragments at pH 5-6. DNase I "footprinting" analysis showed that the transposase protects an inner 25-base-pair region of the 38-base-pair terminal inverted repeat sequence of Tn3. This protection is not dependent on pH. Interestingly, binding of the transposase to the inverted repeat sequences facilitates DNase I to nick at the end of the Tn3 sequence. It was also observed that the transposase protects the end regions of restriction fragments with a cohesive sequence at their 5' end or with a flush end from DNase I cleavage. The specific and nonspecific binding of transposase to DNA is ATP-independent.

Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites.

Transposition of the E. coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins. The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites. The organization of these binding sites on each end, however, is different. Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase. Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu. Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu. The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone.

ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats.

Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites. Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition. We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner. The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin. Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge. Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.