The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli

Abstract

A mutation in the rpoA gene (which encodes the ␣ subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the ؊35 region of the metE promoter that interacts with the ␣ subunit. Our data indicate that the glutamic acid at position 261 in the ␣ subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.The efficiency with which transcription is initiated by RNA polymerase depends on the intrinsic strength of the promoter as well as on the influence of trans-acting regulatory factors. Positive control during transcription initiation is a common mechanism for the regulation of gene expression (23,28). Although a large number of trans-acting regulatory proteins and their binding sites on DNA have been identified (25, 28), relatively little is known about their mechanisms of action. The involvement of direct protein-protein contacts between RNA polymerase and transcription factors has been proposed to explain transcriptional activation (27, 29), though we have limited knowledge of the mechanism involved or the sites of contact, especially in RNA polymerase. Recently, several groups have reported that the sites of interaction of a number of activators with RNA polymerase are localized in its ␣ subunit, which is encoded by the rpoA gene, specifically in the carboxy-terminal region (13-16, 32, 34, 43). Studies indicate that the amino-terminal two-thirds of the ␣ subunit is sufficient for the formation of active enzyme molecules (9,11,12). More recently there have been reports that the ␣ subunit may also make contacts with DNA to activate transcription (1, 30). An AT-rich sequence located upstream of the Ϫ35 region of the Escherichia coli rRNA promoter rrnB P1 stimulates transcription in the absence of any accessory proteins, and mutations in the carboxy-terminal region of the ␣ subunit prevent this stimulation (30).The DNA-binding protein MetR belongs to the LysR family of bacterial activator proteins (10, 31) and is required for the activation of a number of methionine biosynthetic genes in E. coli and Salmonella typhimurium (4,8,21,22,39). For two of the genes, metE and metH, the MetR binding sites required for activation were defined genetically and biochemically and lie just upstream of the RNA polymerase binding site (3,22,38,42). It is possible that...

An intersubunit contact stimulating transcription initiation byE. coliRNA polymerase: interaction of the α C-terminal domain and ς region 4

“…UP elements are present in promoters transcribing rRNAs, tRNAs, and mRNAs, and good proximal subsites are more frequent than good distal subsites or good full UP elements in E. coli promoters (Estrem et al 1999). In this regard, it has been reported that substitutions for E261 reduce factor-independent transcription from the metE promoter in vitro and in vivo (Jafri et al 1995(Jafri et al , 1996. The same ␣CTD-interaction that facilitates UP element proximal subsite function apparently also plays a role in activation by transcription factors at Class I promoters, where ␣CTD is at the same position relative to as at promoters with proximal subsites (Busby and Ebright 1999).…”

“…␣ E261A reduces Class I CRP-dependent activation of the lac promoter Savery et al 2002) and Class I TyrR-dependent activation of the mtr promoter (Yang et al 1997). E. coli strains haploid for E261K also display a variety of other phenotypes (Jafri et al 1995(Jafri et al , 1996, suggesting that this surface on ␣CTD plays a role in multiple cell functions.…”

Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH : Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

“…To determine whether the Strep-tagged ␣ is also functional, we tested for the ability of Strep-tagged ␣ to complement a known rpoA mutant in vivo. The mutant strain GS1040 carries the chromosomal E261K rpoA allele, which prevents growth on glucose minimal medium (16). Transformants of GS1040 carrying the pREII-Strep␣ plasmid exhibit growth on glucose minimal medium indistinguishable from that of the wild-type strain (GS162) or from that of transformants of GS1040 carrying pREII␣, suggesting that Streptagged ␣ is incorporated into RNAP and that the Strep tag does not interfere with function.…”

“…Aliquots of reconstituted core RNAP were incubated in the presence of a 1.5 molar excess of purified 70 subunit to ensure the formation of RNAP holoenzyme for use in single-round runoff transcription assays in the presence of heparin as previously described (16). The activity of each reconstituted RNAP was normalized based on the amounts of lacUV5 and RNA-I transcripts produced when the supercoiled plasmid pRLG593 was used as the template (35).…”