Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter

Abstract

The rightward regulatory region of bacteriophage lambda contains two promoters, p RM and p R , which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A؉T)-rich region (ATR) within the sequence from ؊40 to ؊60 where in the rrnB P1 promoter a stretch of 20 (A؉T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of p RM 's ATR. We determined the effects on RNA polymerase-p RM promoter interaction both of (G؉C) substitutions in the ATR and of amino acid substitutions in the ␣ subunit, known to affect the upstream interaction. We find small (two-to threefold) effects of selected mutations in the ␣ subunit on open complex formation at p RM . However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by ( The rightward regulatory region of bacteriophage lambda contains two promoters, p R and p RM , which are divergently transcribed from start sites separated by only 82 bp (25). Open complex formation at the p R promoter is much faster than at p RM ; therefore, the vast majority of RNA polymerases (RNAPs) forming an open complex at the latter promoter do so on a fragment that already contains a polymerase at p R . It was found by us (14, 15) as well as by Gussin's group (6, 28) that mutations which inactivate the p R promoter facilitate open complex formation at p RM . These results suggest that p R -bound RNAP interferes with open complex formation at p RM . Surprisingly, a 10-bp deletion between the two promoters appears to abolish the interference (22), as it results in increased activity of p RM whether p R is inactivated by mutation or not.The identity of promoters for Escherichia coli RNAP is mostly determined by two DNA elements, the Ϫ10 and Ϫ35 regions (21). The ability to recognize these two regions is imparted upon the RNAP holoenzyme by its subunit (12). The promoters for E. coli rRNA (27), as well as some other strong promoters in E. coli (17)(18)(19) and Bacillus subtilis (2, 13), have been found to additionally have (AϩT)-rich regions (ATRs) upstream of bp Ϫ40. These so-called UP elements (27) are typically 20 bp in length. Transcription in vivo and in vitro (27) as well as the rate of open complex formation at the rrnB P1 promoter (26) was decreased by more than 10-fold because of deletion of this UP element. The C-terminal domain (amino acids 249 to 329 [3,23]) of the ␣ subunit of RNAP has been implicated in UP element recognition: deletion of this domain greatly impaired the ability of RNAP to respond to the UP element both in vivo and in vitro (27). In subsequent studies, amino acid substitutions in the ␣ subunit were employed to pinpoint the amino acids involved in the interaction with the UP element to those at positions 262 to 269 and 296 to 299 (8). Recent determinations of the structure of the Cterminal domain of the ␣ subunit show that the location of these amino acids is compatible wit...

Structural Modules of RNA Polymerase Required for Transcription from Promoters Containing Downstream Basal Promoter Element GGGA

“…In contrast, termination efficiency of T. aquaticus RNAP did not depend on temperature within the range studied ( Fig. 1, lower panel, lanes [1][2][3][4]. The result may indicate that at high temperatures, elongation complexes of T. aquaticus RNAP are more stable than E. coli RNAP complexes.…”

“…Presently, no systematic analysis of upstream promoter sequences of T. aquaticus and related bacteria have been performed, and the specificity of DNA recognition by T. aquaticus ␣CTD remains unknown. Because the deletion of ␣CTDs impaired recognition of T7 A1 and sTap2 promoters, as well as other sTap-based promoters with varying upstream sequences, 4 we propose that at least on these promoters, T. aquaticus ␣CTDs stimulate RNAP binding through favorable but sequence-nonspecific interactions with DNA. Nonspecific contacts of E. coli RNAP ␣CTDs with upstream DNA have been demonstrated to stimulate transcription from promoters that lack the UP element by increasing the rate of RNAP-promoter association (5,6).…”

Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter

“…(19) showed that the ␣-subunit of RNA polymerase can also interact with an AT-rich sequence (UP element) which is located upstream of the Ϫ35 region of the rrnBp 1 promoter of E. coli and is responsible for enhancement of transcription initiation. Such UP elements appear to be a feature of many promoters (8,19,22).The TyrR protein of E. coli is both a repressor and an activator and controls the expression of a group of eight transcriptional units (TyrR regulon) whose translational products are involved in the biosynthesis or uptake of the three aromatic amino acids (17). The TyrR protein contains three structural domains (5), and genetic analysis has mapped the activation function of TyrR to the N-terminal domain (4,24,25).…”

“…Work by Ross et al (19) showed that the ␣-subunit of RNA polymerase can also interact with an AT-rich sequence (UP element) which is located upstream of the Ϫ35 region of the rrnBp 1 promoter of E. coli and is responsible for enhancement of transcription initiation. Such UP elements appear to be a feature of many promoters (8,19,22).…”

Promoter Interference in a Bacteriophage Lambda Control Region: Effects of a Range of Interpromoter Distances

“…Therefore, for the wild-type control region, in vitro RNA polymerase (RNAP)-p RM interactions occur almost exclusively in the context of another RNAP already bound to p R . It has been previously shown that this p R -bound RNAP interferes with open complex formation at p RM (16,17,21,34,37). The effect is not exerted at the initial binding of RNAP to the promoter but rather at a subsequent step (16, 34) that is likely a conformational change in the RNAP (9).…”