TRANSPOSITION AND REGULATION OF TRANSPOSITION IN THE TRANSPOSON TN3 (TRANSPOSASE, DNA BINDING, ATP-DEPENDENT).

摘要

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, 5000 bp in length, which contains three genes: (i) the tnpA gene, which codes for the transposase protein required for transposition, (ii) the tnpR gene, which codes for the repressor protein which regulates transposition and also acts as a recombinase during the transposition process, and (iii) the bla gene, which codes for the beta-lactamase protein which confers antibiotic resistance.; This dissertation focuses upon the molecular mechanism of transposition and regulation of transposition in the transposon Tn3. The results of three distinct but related lines of investigation are reported including: (i) an analysis of the E. coli RNA polymerase binding sites and transcription initiation sites in the Tn3 transposon, (ii) an analysis of plasmid cointegrates and their resolution mediated by transposon Tn3 mutants, and (iii) an analysis of the ATP dependent site-specific DNA binding activity associated with purified Tn3 transposase enzyme.; Two RNA polymerase binding sites were identified in Tn3. One, at the amino terminus of the beta-lactamase gene, in which the initiation site for the beta-lactamase mRNA was identified, and the second, located between the coding frames for the transposase and repressor proteins, which contains the transcription initiation sites for the transposase and repressor mRNAs. A model is presented for transcriptional regulation of this divergently transcribed promoter region by the Tn3 repressor protein.; An analysis of cointegrate plasmids formed by mutants of the transposon Tn3 confirmed that cointegrates are intermediates in the transposition process in that they contain two complete copies of Tn3 flanked by 5 base pair duplication at the site of insertion. Furthermore, cointegrates formed by repressor mutants could be resolved into normal products of transposition by complementation with wild type Tn3 repressor protein.; The Tn3 transposase protein, purified from a strain which overproduces the protein was shown to possess an ATP dependent site-specific DNA binding activity. The transposase is capable of binding double stranded DNA nonspecifically in the absence of ATP. This nonspecific DNA binding is sensitive to heparin challenge. However, in the presence of ATP, DNA binding specific for the ends of the transposon is observed and this specific DNA binding is resistant to heparin challenge. A molecular model for the Tn3 transposase ATP dependent site-specific DNA binding activity is presented.