Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.788541
Title: Studies on the tnsA and tnsB genes of transposon Tn7
Author: Gawthrop, Angela Barbara
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Abstract:
Tn7 is a 14kb transposable element found in a variety of enteric bacteria. It encodes five genes, tnsA, tnsB, tnsC, tnsD and tnsE which are required for transposition of the element. Two of these genes, tnsA and tnsB, are the subject of this study. The tnsA gene is poorly expressed from the Tn7 tns promoter and ribosome binding site; the possible reasons for this were investigated, through both examination of the DNA sequence and experimentation. Different expression systems for tnsA were utilised, and use of the pT7-7 translational fusion expression vector was found to yield high concentrations of the TnsA protein. This protein was, however highly insoluble. Various strategies for solubilising the protein were tested, and conditions under which partially purified TnsA could be maintained in a soluble form were found. Regulation of transposition of a number of prokaryotic elements is brought about by the binding to the terminal repeat sequences of an inactive truncated form of the transposase protein. TnsB was previously known to bind to repeated 22bp motifs at the Tn7 ends; peptides believed to be proteolytic fragments of TnsB are produced in vivo particularly under certain growth conditions, and it was hypothesised that these might be involved in regulation of Tn7 transposition. A series of plasmids encoding TnsB peptides truncated at the amino or carboxyl end was therefore constructed. These plasmids were used in three different lines of investigation: in vitro binding of protein extracts derived from cells expressing these truncated tnsB genes to DNA fragments from the left and right ends of Tn 7; galactokinase assays to measure the degree of repression of the tns promoter (which overlaps the terminal repeat at the Tn7 right end) in the presence of the TnsB peptides; and in vivo transposition frequencies of Tn7 in the presence of a high concentration of TnsB peptide, relative to that of the wild type TnsB. Results suggest that, although binding is observed which is specific to TnsB peptides including a region near the amino end, these peptides do not significantly repress transcription of the tns genes or reduce the observed in vivo transposition frequencies. During the course of this work, a weak promoter was identified in the tns A coding sequence and reading in the opposite direction to the tns genes; it is thought unlikely that this plays a significant role in regulation of transposition. A sequence bearing similarity to that of a helix-turn-helix DNA binding motif had been previously identified towards the amino end of TnsB. All the TnsB peptides shown here to exhibit binding activity to the right or left end motifs of Tn7 contained this sequence. Site-directed mutagenesis of the putative recognition helix of TnsB did not, however, lead to loss of binding activity, and so the existence of the helix-tum-helix remains in doubt. Agarose and polyacrylamide binding gels were used to search for synapsis of the Tn7 ends mediated by TnsB; such a synapsis is likely to be a prerequisite for transposition. Results from polyacrylamide gel electrophoresis were consistent with the formation of DNA-protein-DNA complexes involving linear DNA fragments. TnsB-specific retardation was also observed on agarose gels with circular molecules containing a mini-Tn7, but there was no evidence that this was due to synapsis of the Tn7 ends and not simply to the added mass of the binding protein molecules. The clarity of the stained gels would be improved by the use of pure TnsB protein. Strategies for improving on the previous partial purification procedure were therefore investigated but no significant improvement was made; insolubility of TnsB in low salt concentrations and a predilection for proteolysis (the latter of which might be solved by carrying out the whole procedure at low temperature) were the main problems encountered.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.788541  DOI: Not available
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