Expression and Function of Tn7 Transposition Proteins

[摘要] Transcriptional and translational fusion vectors were used to identify and characterise transcriptional control signals and translated reading frames in the transposition region of the transposon Tn7. Two promoters, one strong, P1, and one much weaker, P2, were detected and their precise levels of transcription were measured. Five proteins, corresponding to the tnsA, tnsB. tnsC, tnsD, and tnsE genes on the complementation map, were also identified. All but one of these proteins were expressed in low amounts. Transcription and translation of all five genes appeared to proceed in the same direction. The control of transcription was studied by placing fragments expressing Tn7 transposition functions in trans with the major Tn7 promoter, P1, and examining the effects on transcription. It was found that the presence of the tnsB gene product in trans with P1, resulted in a considerable decrease in P1 activity. The binding properties of the tnsB and tnsD gene products were examined employing a gel electrophoresis protein-DNA binding assay. In the absence of purified Tn7 proteins, whole-cell crude extracts from overproducing strains were used. It was found that tnsB, a protein required for both modes of Tn7 transposition, recognises and binds specifically to sequences in the right end of the element. Preliminary results suggested that tnsD, the "hot site" specific protein, binds to the chromosomal attachment site ("hot site"). The implications of these events on the mechanism of transposition were discussed. The role of DNA adenine methylase (dam protein) in Tn7 transposition was investigated. The existence of a GATC (dam sensitive site) in a critical region in the element (within the repeats at the right end terminus) and our belief that Tn7 transposes via a conservative mechanism, promoted this investigation. Tn7 transposition was monitored in unmethylated, hemimethylated and fully methylated environments. No significant difference in the transposition levels in any of these three backgrounds was observed. This seemed to suggest that Tn7 transposition is not regulated by dam methylation, however, alternative interpretations were discussed.

[发布机构] University:University of Glasgow

[关键词] Genetics, Molecular biology