A DNA sequence rich in (A+T), located upstream of the -10, -35 region of the Escherichia coli ribosomal RNA promoter rrnB P1 and called the UP element, stimulates transcription by a factor of 30 in vivo, as well as in vitro in the absence of protein factors other than RNA polymerase (RNAP). When fused to other promoters, such as lacUV5, the UP element also stimulates transcription, indicating that it is a separate promoter module. Mutations in the carboxyl-terminal region of the alpha subunit of RNAP prevent stimulation of these promoters by the UP element although the mutant enzymes are effective in transcribing the "core" promoters (those lacking the UP element). Protection of UP element DNA by the mutant RNAPs is severely reduced in footprinting experiments, suggesting that the selective decrease in transcription might result from defective interactions between alpha and the UP element. Purified alpha binds specifically to the UP element, confirming that alpha acts directly in promoter recognition. Transcription of three other promoters was also reduced by the COOH-terminal alpha mutations. These results suggest that UP elements comprise a third promoter recognition region (in addition to the -10, -35 recognition hexamers, which interact with the sigma subunit) and may account for the presence of (A+T)-rich DNA upstream of many prokaryotic promoters. Since the same alpha mutations also block activation by some transcription factors, mechanisms of promoter stimulation by upstream DNA elements and positive control by certain transcription factors may be related.

No abstract

An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20‐ to 30‐fold over that of the same promoter lacking the UAR. We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10‐ to 20‐fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1‐lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5‐fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5‐fold in a Fis‐ strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.

Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp͞ DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.RNA polymerase ͉ rRNA transcription ͉ transcription initiation ͉ amino acid biosynthesis ͉ stringent response A mino acid starvation in Escherichia coli results in global changes in gene expression called the ''stringent response'' (reviewed in ref. 1). This regulatory response is initiated by entry of uncharged tRNAs into the A site of the ribosome and activation of the ribosome-associated RelA protein to synthesize the ''alarmone'' ppGpp (used here to refer to both guanosine 5Ј-diphosphate 3Ј-diphosphate and its pentaphosphate precursor). During starvation for amino acids, expression of stable RNA (rRNA and tRNA) is inhibited, whereas expression of enzymes for amino acid biosynthesis and transport is induced. These negative and positive responses are both relA dependent (e.g., refs. 2-6).ppGpp directly and specifically inhibits the initiation of transcription from stable RNA promoters in vivo and in vitro (7)(8)(9)(10)(11)(12). Although the details of the mechanism by which ppGpp exerts its effects on transcription initiation are still ill-defined, it has been shown that ppGpp decreases the lifetimes of competitorresistant complexes between RNA polymerase (RNAP) and all promoters that have been examined (12). The complex formed by RNAP at rRNA promoters is intrinsically short-lived, and ppGpp further decreases the lifetime of this complex. Thus, we have proposed that ppGpp shifts the equilibriu...

No abstract