Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς ⁷⁰

Abstract

Amino acid substitutions in Escherichia coli 70 were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.Core RNA polymerase (␣ 2 ␤␤Ј) requires the variable specificity subunit, sigma ( ), to direct promoter-dependent transcription (1,3,4,12,18,22,23,26). Following promoter binding, holoenzyme (␣ 2 ␤␤Ј ) progresses through several intermediate complexes, en route to a stable initiated open complex (2,14).factor has been implicated in stages of initiation beyond promoter recognition (8,9,13,15,17). Recently, we showed that the conserved amino terminal domain (region 1) of Escherichia coli 70 is important for the process of strand melting and initiated complex formation at the p R promoter (24).Region 1 is unique to the primary factors, yet little is known of its function. Deletion of region 1.1 (amino acids 1 to 100) from 70 has two major consequences for holoenzyme. The first is inefficient progression from the closed to the strand-separated open complex. This can be overcome by increasing the time allowed for formation of holoenzyme-promoter complexes and is lessened by addition of region 1.1 in trans. The second and more deleterious effect is impaired transition from the strand-separated open complex to a stable initiated complex (RP init D]), as well as a high degree of acidity (40%) within the segment from amino acids 50 to 75 (24). Here, we test whether alterations at these conserved positions or in the overall acidity of the region influence initiation by holoenzyme (E ).Site-directed mutagenesis (10) and the Expand high fidelity PCR system (Boehringer Mannheim) were used to create substitutions at positions 52, 53, 55, 61, 57, 58, 63, 64, and 69 (Table 1). rpoD was mutagenized in M13 phage (10) and amplified with oligonucleotides that incorporated restriction sites at the 5Ј and 3Ј ends of the fragment. The restricted fragments were ligated into pQE30-T (24). PCR mutagenesis was used to amplify a fragment corresponding to the 3Ј end of the rpoD gene with a 5Ј mutagenic oligonucleotide and a 3Ј oligonucleotide that incorporated a restriction site. A concurrent round of amplification included a 5Ј oligonucleotide complementary to the 5Ј end of rpoD and a 3Ј oligonucleotide with complementarity to an internal segment of rpoD, downstream from the genetic alteration(s). The 5Ј and 3Ј PCR fragments were mixed, and the full-length mutagenized rpoD gene was amplified, gel isolated, digested, ligated into pQE30-T, transformed into E. coli XL1 Blue (Stratagene), and sequenced to confirm the changes. The plasmids were transformed into E. coli 19284 (rpoD800, W3110 srl::Tn10 recA lacI q ) to test for function in vivo (24). Transformation mixtures were split and spread onto Luria-Bertani plates containing ampicillin (100 mg/ml), kanamycin (30 mg/ml), and 2% glucose and then inc...

Formation of Intermediate Transcription Initiation Complexes at p fliD and p flgM by ς 28 RNA Polymerase

“…Unexpectedly, the presence of the first three NTPs (ATP, CTP, and GTP) allowed the E 28 -pflgM complex to remain stable during a challenge with 50 g of heparin per ml (Fig. 7, lanes 6 to 8) 4 and piperidine. DNA alone was treated at 37°C.…”

“…Region 1.1 is found only in primary factors (37) and prevents the subunit from binding to DNA in the absence of the core subunits (15). Deletion of region 1.1 of 70 results in defective promoter melting and transcription initiation at the P R promoter (65), while single amino acid substitutions have been shown to affect initial DNA binding by holoenzyme (3) and the stability of 70 (4). Region 1.2, while highly conserved and found in almost all factors, has not yet been assigned a function (37).…”

Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation

“…S1). In addition to the proposed functions in preventing interactions of free with DNA, region 1.1 likely plays an important role in holoenzyme assembly and open complex formation (25)(26)(27)(28). Several studied amino acid substitutions and deletions in this region in Eco 70 (supplemental Fig.…”

“…Several studied amino acid substitutions and deletions in this region in Eco 70 (supplemental Fig. S1) decreased RNAP activity, impaired open complex formation, and/or decreased the stability of promoter complexes (25,(27)(28)(29).…”

In Vitro Properties of RpoS (σ S ) Mutants of Escherichia coli with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted

“…Subregion 1.1 in E. coli 70 (comprising the segment from approximately residues 30 through 100) has been ascribed various functions and activities, including those of masking the DNA-binding domain(s) in free (10,11) and mediating the binding of with core enzyme (16,32), thereby influencing promoter binding and the efficiency of transcription initiation by holoenzyme (4,5,13,31,46,47). Deletion of subregion 1.1 of 70 is associated with a dominant lethal phenotype in E. coli (10,38).…”