Kinetic studies of the modulation of ada promoter activity by upstream elements.

Abstract

We have determined the kinetics of initiation of transcription of the wild‐type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M‐1 and 1.7 x 10(‐2) s‐1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild‐type sequence with an up‐mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (‐0.05), leading to a 12‐fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)

Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium

“…DNA curvature has been observed or predicted from sequence data in the upstream regions of a number of promoters, where it is believed to play an important role in increasing transcription rates. Sequences upstream of the -35 regions of such promoters generally contain phased tracts of A, or Tn (2,4,27,36,47), which are known to favor intrinsic DNA bending (29). In addition, induced DNA bending is thought to be a significant factor in the mechanism of a number of transcriptional activators (46,59), of which the most extensively studied is the E. coli catabolite gene activator protein (CAP) (reviewed in reference 28).…”

Intrinsic DNA Distortion of the Bacteriophage MumomP1 Promoter Is a Negative Regulator of Its Transcription

“…UP elements, when present, are integral components of promoters, because they interact with the carboxy terminal domain of the RNAP ␣-subunit (12). In vitro studies showed that the E. coli RNAP ( 70 ) holoenzyme alone is sufficient for transcriptional activity from several such promoters (12,14,15).…”

Conformational properties of the ?35 region of the trp promoter in solution: comparison of the wild-type sequence with an AT transversion

“…These are the -35 region, having the consensus sequence TTGACA, a spacer of 17_+2 base pairs, and the -10 region, or Pribnow box, which has the consensus sequence TATAAT (Harley and Reynolds 1987). In some promoters, there is a fourth AT-rich stretch downstream of the --35 region which may be involved in bending the promoter around RNA polymerase (Bertrand-Burggraff et al 1990), or other sites which are recognised by transcriptional activators such as the catabolite activator protein (de Crombrugghe et al 1984).…”