The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

Abstract

The Escherichia coli guaB promoter (P guaB ) regulates the transcription of two genes, guaB and guaA, that are required for de novo synthesis of GMP, a precursor for the synthesis of guanine nucleoside triphosphates. The activity of P guaB is subject to growth rate-dependent control (GRDC). Here we show that the A؉T-rich sequence located between positions ؊59 and ؊38 relative to the guaB transcription start site stimulates transcription from P guaB ϳ8-to 10-fold and, in common with other UP elements, requires the C-terminal domain of the RNA polymerase ␣ subunit for activity. Like the rrnB P1 UP element, the P guaB UP element contains two independently acting subsites located at positions ؊59 to ؊47 and ؊46 to ؊38 and can stimulate transcription when placed upstream of the lacP1 promoter. We reveal a novel role for the P guaB UP element by demonstrating that it is required for GRDC. The involvement of the UP element in GRDC also requires the participation of sequences located at least 100 bp upstream of the guaB transcription start site. These sequences are required for down-regulation of P guaB activity at lower growth rates.Escherichia coli RNA polymerase (RNAP) is a multisubunit complex consisting of an ␣ subunit homodimer, single ␤, ␤Ј, and subunits, and one of several subunits (14). The ␣ subunit contains an N-terminal domain (␣NTD; residues 8 to 232) and a C-terminal domain (␣CTD; residues 249 to 329) (5, 21, 44). ␣NTD and ␣CTD are separated by a flexible linker that is at least 13 amino acids long (5,20,25). ␣CTD plays important roles in transcription activation by contacting a number of transcription factors (6, 18) or through interacting with an AϩT-rich DNA sequence referred to as the UP element (16).UP elements stimulate transcription by recruiting ␣CTD to DNA (32, 43). The best-studied UP element spans the region from Ϫ59 to Ϫ38 at the rrnB P1 promoter (16). The rrnB P1 UP element contains two domains, a ϳ9-bp proximal UP element subsite located at positions Ϫ46 to Ϫ38 and a ϳ13-bp distal UP element subsite located at positions Ϫ59 to Ϫ47. Each subsite functions independently by recruiting one ␣CTD (11). The amino acid residues in ␣CTD that comprise the 265 determinant, including residues V264, R265, N268, N294, G296, K298, and S299, interact with UP element and A-tract sequences by contacting the DNA backbone, and R265 makes additional contacts with bases in the minor groove (4, 32, 43). These residues are the most important for rrnB P1 UP element utilization both in vivo and in vitro (13, 35). In the absence of a strong distal UP element subsite, ␣CTD bound to a consensus or near-consensus proximal UP element subsite also makes productive contacts with region 4.2 of 70 that serve to stimulate transcription (7,35).The E. coli guaB promoter (P guaB ) regulates the transcription of two genes, guaB and guaA, which together constitute the guaBA operon. The guaB and guaA genes encode IMP dehydrogenase and GMP synthetase, respectively, and are required for the biosynthesis of GMP from IMP (22, 42). P guaB...

Downregulation of the Escherichia coli guaB Promoter by Upstream-Bound Cyclic AMP Receptor Protein

“…VOL. 191, 2009 REGULATION OF THE E. COLI guaB PROMOTER 6095 scription reactions were performed as previously described, in the presence or absence of CRP, except that KCl was used at a concentration of 100 mM (33). Reactions were terminated with an equal volume of stop solution (95% deionized formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol).…”

“…Cells from overnight cultures grown in medium supporting the lowest growth rate were inoculated to a starting optical density at 600 nm (OD 600 ) of 0.02 into different media that supported a range of growth rates and were grown with aeration at 37°C. The set of culture media used for growing strains containing wild-type crp and cyaA alleles was based on M9 minimal medium and is referred to here as standard media (33). The set of media used to grow strains containing deletions in the crp and/or cyaA gene is referred to as CRP media and was based on M9 minimal medium containing one of the following (in order of increasing growth rates supported for wild-type strains): 0.4% (wt/vol) fructose, 0.4% (wt/vol) glucose, 0.4% (wt/vol) fructose plus 20 amino acids, 0.4% (wt/vol) fructose plus 1% (wt/vol) Casamino Acids, 0.4% (wt/vol) glucose plus 20 amino acids, or 0.4% (wt/vol) glucose plus 0.8% (wt/vol) Casamino Acids.…”

“…An UP element, located immediately upstream of the promoter Ϫ35 region, strongly enhances transcription and is required for GRDC of this promoter (28,33). Three binding sites for the nucleoid-associated protein FIS have been identified, centered near positions Ϫ11, ϩ8, and ϩ29 relative to the guaB transcription start site.…”

“…In a separate transcriptomic study, guaB was identified as one of a number of genes that are subject to "CRP-dependent glucose activation" (26). We have previously shown that sequences located between positions Ϫ133 and Ϫ100, which include the putative CRP site, are required for GRDC of P guaB (33). To resolve the apparent inconsistencies between some of these observations, we carried out an investigation into the role of CRP at P guaB .…”

“…P guaB responds to a variety of physiological signals. For example, the activity of P guaB increases as a function of the cellular growth rate, such that guaBA mRNA forms an increasing fraction of total cell mass at higher growth rates (16,33,34). This form of regulation is referred to as growth rate-dependent control (GRDC) (17, 27).…”