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N L Craig

Publications and source records attributed to N L Craig.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA.

The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-gamma-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.

Adenosine Diphosphate↗
PubMed1991

Purification of TnsB, a transposition protein that binds to the ends of Tn7.

We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7. The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein. The apparent molecular mass of denatured TnsB protein is approximately 85 kDa. Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape. Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon. The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition. Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7. These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.

Amino Acid Sequence↗
PubMed1991

Interaction of the Tn7-encoded transposition protein TnsB with the ends of the transposon.

We have used several high resolution methods to examine the interaction of TnsB, a transposition protein encoded by the bacterial transposon Tn7, with its binding sites at the ends of the transposon. These binding sites lie within the DNA segments that are directly involved in transposition. We show that the binding of TnsB to DNA can promote DNA bending, suggesting that the interaction of TnsB with the ends may result in formation of a highly organized protein-DNA complex. We also identify likely positions of close contact between of TnsB and its binding sites. Analysis of the interaction of TnsB with intact Tn7 ends reveals TnsB occupies its binding sites in a particular order, the sites immediately adjacent to the transposon termini being occupied only after other inner sites are bound. Such ordered occupancy suggests that the various binding sites have differing apparent affinities for TnsB.

Alkylation↗
PubMed1991

Identification of transposition proteins encoded by the bacterial transposon Tn7.

The bacterial transposon, Tn7, encodes an elaborate array of transposition genes, tnsABCDE. We report here the direct identification of the TnsA, TnsB, TnsC and TnsD polypeptides by immunoblotting. Our results demonstrate that the complexity of the protein information devoted to Tn7 transposition is considerable: the aggregate molecular size of the five Tns polypeptides is about 300 kDa. We also report the sequence of the tnsA gene and of the 5' ends of tnsB and tnsD. This analysis reveals that all five tns genes are oriented in the same direction within Tn7.

Amino Acid Sequence↗
PubMed1991

Tn7 transposition in vitro proceeds through an excised transposon intermediate generated by staggered breaks in DNA.

We have developed a cell-free system in which the bacterial transposon Tn7 inserts at high frequency into its preferred target site in the Escherichia coli chromosome, attTn7; Tn7 transposition in vitro requires ATP and Tn7-encoded proteins. Tn7 transposes via a cut and paste mechanism in which the element is excised from the donor DNA by staggered double-strand breaks and then inserted into attTn7 by the joining of 3' transposon ends to 5' target ends. Neither recombination intermediates nor products are observed in the absence of any protein component or DNA substrate. Thus, we suggest that Tn7 transposition occurs in a nucleoprotein complex containing several proteins and the substrate DNAs and that recognition of attTn7 within this complex provokes strand cleavages at the Tn7 ends.

Adenosine Triphosphate↗
PubMed1991

Tn7: a target site-specific transposon.

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.

Bacterial Proteins↗
PubMed1989

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity.

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.

Base Composition↗
PubMed1989

Tn7 transposition: recognition of the attTn7 target sequence.

The bacterial transposon Tn7 encodes two distinct but overlapping transposition pathways. tnsABC + tnsD promote transposition to a specific site, attTn7, while tnsABC + tnsE promote transposition to many other sites unrelated to attTn7. We have identified a tnsD-dependent DNA binding activity that specifically recognizes attTn7. We have localized the recognition sequences for this activity to a 28-base-pair region and have shown that this same region can provide specific properties of an attTn7 target in vivo. Interestingly, these sequences are positioned more than 25 base pairs from the specific point of Tn7 insertion.

Base Sequence↗
PubMed1988

Tn7 transposition: two transposition pathways directed by five Tn7-encoded genes.

The bacterial transposon Tn7 is capable of high-frequency transposition to a specific site in the Escherichia coli chromosome, attTn7, and of low-frequency transposition to sites other than attTn7. Using an in vitro insertional mutagenesis procedure, we have identified and characterized five tns (Tn seven) genes that are essential for Tn7 transposition. Three of these genes, tnsA, tnsB, and tnsC, are required, but are not sufficient, for all Tn7 transposition events. In addition, tnsD is specifically required for transposition to attTn7, whereas tnsE is specifically required for transposition to other sites. Thus, Tn7 is an elaborate transposon that encodes two distinct but overlapping transposition pathways.

DNA Transposable Elements↗
PubMed1988

Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion.

Transposon Tn7 transposes at high frequency to a specific site, attTn7, in the Escherichia coli chromosome. We devised a quantitative assay for Tn7 transposition in which Tn7-end derivatives containing the cis-acting transposition sequences of Tn7 transpose from a bacteriophage lambda vector upon infection into cells containing the Tn7-encoded transposition proteins. We used this assay to identify a 68-base-pair DNA segment containing the sequences essential for attTn7 target activity. This segment is positioned asymmetrically with respect to the specific point of Tn7 insertion in attTn7 and lacks obvious homology to the sequences at the ends of Tn7 which participate directly in transposition. We also show that some sequences essential for attTn7 target activity are contained within the protein-coding sequence of a bacterial gene.

Base Sequence↗
PubMed1988

Recognition of Escherichia coli attTn7 by transposon Tn7: lack of specific sequence requirements at the point of Tn7 insertion.

Transposon Tn7 inserts at high frequency into a specific site in the Escherichia coli chromosome called attTn7. We show that the point of Tn7 insertion in attTn7 lies within the transcriptional terminator of the bacterial glmS gene. We have exploited the glmS transcription terminator to isolate mutants with altered sequences at the point of Tn7 insertion and have used these mutants to show that the nucleotide sequence at the point of Tn7 insertion is irrelevant to attTn7 target activity. Thus, the nucleotides which provide attTn7 target activity are distinct from the point of Tn7 insertion. We have also examined the effect of transcription on the capacity of attTn7 to act as a target for Tn7 transposition. Our results suggest that transcription of attTn7 does not modulate its Tn7 target activity.

Base Sequence↗
PubMed1987

Identification of a transposon Tn7-dependent DNA-binding activity that recognizes the ends of Tn7.

The bacterial transposon Tn7 is distinguished by its capacity for high-frequency transposition to a specific site in the Escherichia coli chromosome. tnsB is one of the five Tn7-encoded transposition genes. We have identified in vitro a tnsB-dependent DNA binding activity that interacts specifically with cis-acting transposition sequences at the Tn7 termini. Although the left and right termini of Tn7 are structurally distinct, each end contains several copies of a closely homologous 22-base-pair sequence. We present results indicating that this 22-base-pair repeat sequence is recognized by the tnsB-dependent binding activity.

Bacterial Proteins↗
PubMed1978

Escherichia coli recA gene product inactivates phage lambda repressor.

Phage lambda repressor is inactivated and cleaved into two detectable fragments during incubation with purified Escherichia coli recA gene protein in vitro, in a reaction that requires ATP. This reaction reproduces the recA-dependent inactivation of repressor that occurs in vivo during induction of the SOS functions. The proteolytic activity may reside in the recA protein itself and may be a fundamental activity of it.

Adenosine Triphosphate↗