In vivo and in vitro effect of mutations in tetA promoter from pSC101: insertion of poly(dA.dT) stretch in the spacer region does not inactivate the promoter

Abstract

Two mutants, mapping at the HindIII site (between the consensus sequences) of the pSC101 tetA promoter, were studied: MA2 corresponds to a 4 bp deletion between positions -12 and -15; B30 bears a 44 bp insertion C(TA)21 G at the HindIII site. Both mutants were assayed in vivo (ability of the plasmid to confer resistance to tetracycline, plasmid-directed protein synthesis, S1-mapping of mRNA) and in vitro (abortive initiation assay). Compared to w.t., MA2 is a poor promoter in vivo; RNA polymerase binding, complex activation and rate of initial oligonucleotide synthesis are strongly reduced in vitro; this is in keeping with the known effects of altering the consensus elements in E. coli promoters. In contrast, B30 shows in vivo a promoter activity only slightly reduced in comparison to that of the w.t. tetA promoter; both in vivo and in vitro, the transcription start site is outside and downstream the (TA)21 stretch, 5-7 bp upstream that found in the w.t. To adjust the behaviour of B30 and the claimed consensus distance between the E. coli promoter consensus sequences, some structural modification in the (TA)21 stretch -either spontaneous or induced by RNA polymerase- can be hypothesized. Unless the (TA)21 stretch itself plays the role of a relatively good promoter, the results suggest that promoter-specific elements may be distributed along the DNA sequences over distances longer, but seldom less, than the 17 +/- 2 bp consensus distance.