Spacer mutations in the lac ps promoter.

Abstract

Mutations have been constructed that delete either one or two base pairs near position -19 in the lac pS promoter. Deletion of either of two adjacent base pairs increases the rate of open complex formation by nearly an order of magnitude. Two promoters that have different single-base deletions are indistinguishable by either their rates of open complex formation or stability of the open complexes once formed. However, simultaneous deletion of both base pairs produces a promoter that forms complexes at a rate similar to that of the unmodified DNA sequence. The maximal rate of open complex formation is achieved at a spacer length of 17 base pairs, the most frequently occurring spacer length among promoters. These results suggest that the spacing between the two strongly conserved regions of sequence homology is an important determinant of the rate of open complex formation. A model is suggested that proposes that three important promoter elements, the -10 region, the -35 region, and the spacer region, act simultaneously to facilitate open complex formation by RNA polymerase.The promoter is a DNA sequence that directs RNA polymerase to bind and initiate transcription specificially; Comparison of prokaryotic promoter sequences has shown two regions of sequence homology located -10 and 35 base pairs prior to the start point of transcription (1, 2). The precise roles of each of these regions in promoter function are unknown. The importance of these homologies is supported by the concentration of promoter mutations in these sequences. In general, sequence changes that decrease homology to the consensus tend to be down promoter mutations.The region between the -10 and -35 sequences has not been shown to be of importance. These sequences are poorly conserved and few mutations exist in this region. Nevertheless, the number of intervening nucleotides is highly conserved (1). This suggests that proper spacing between the -10 and -35 sequences may be functionally important and prompts us to identify this as a third region of the promoter, the "spacer" region.To test the importance of this conserved spacer length, we have constructed mutations that change the spacer length in the lac pS promoter. This promoter has a spacer region one nucleotide longer than the consensus length. Deletion in vitro yields promoter DNA that is of consensus length and shorter. The properties of these strains lend support to the hypothesis that the spacer region is a third important element in promoter function. MATERIALS AND METHODSRNA polymerase holoenzyme was prepared according to Gonzales et at (3). Nuclease S1 was purchased from Miles and purified on sulfopropyl-Sephadex by a modification of Vogt (4).Bovine serum albumin (fraction V; Pentex) was purified by passage over a heparin agarose column. Other materials were as described (5).For construction of deletion mutants (see Fig. 1), EcoRI restriction fragments containing the ps promoter (5.5 ,ug; ref. 6) were ligated overnight at 15'C. The resultant polymer was cleaved with Hpa II. Ha...

Monomer–dimer control of the ColE1 P cer promoter

“…These results are consistent with many previous observations that maximum promoter activity and open complex formation by RNA polymerase occurs with a spacer length of 17 bp and that lengthening or shortening the spacer leads to a decrease in promoter activity (Berman & Landy, 1979 ;Ackerson & Gralla, 1982 ;Mandecki & Reznikoff, 1982 ;Stefano & Gralla, 1982 ;Aoyama et al, 1983 ;Mandecki et al, 1985). A less consistent observation was that the P cer derivative with a 14 bp spacer showed a 1n9-fold increase in promoter activity relative to wild-type P cer , and was similar to P cer promoter derivatives with a 16 bp spacer.…”

DNA twist as a transcriptional sensor for environmental changes

“…The point that has been generally overlooked is that DNA twist may be an environmental sensor where dynamic changes in the helical pitch of DNA influence promoter activity. There have been allusions to the possibility that there may be promoters which are sensitive to twist (Stefano and Gralla, 1982;O'Neil, 1989), and recently it has been proposed that merR regulatory protein activates transcription of the mer promoter by decreasing twist (Ansari etal., 1992). However, in spite of these earlier suggestions we believe that, with the exception of the mer promoter, the notion of twist-sensitive promoters has been largely unappreciated and that its implications have been undeveloped.…”

Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

“…Interestingly, the lacTp promoter sequence of the class III revertants was exactly the same as that of the functional lacTp promoter described for L. casei BL23 . This reflects the fact that maximum promoter activity and open complex formation by RNA polymerase usually happen with promoters in which the length of spacer between the 235 and 210 boxes is 17 bp (Berman & Landy, 1979;Ackerson & Gralla, 1983;Mandecki & Reznikoff, 1982;Stefano & Gralla, 1982;Aoyama et al, 1983;Mandecki et al, 1985;Chatwin & Summers, 2001). For class IV, V and VI revertants, a point mutation was found in the 210 (TATAAC), 210 (TAAACT) and 235 (TTGACA) boxes of the lacTp promoter, respectively.…”