Changes in conserved region 2 of Escherichia coli σ70 affecting promoter recognition

Waldburger, Carey D.; Gardella, Thomas J.; Wong, Rex; Susskind, Miriam M.

doi:10.1016/s0022-2836(05)80345-6

Cited by 185 publications

(219 citation statements)

References 35 publications

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“…It is clear that the change in 1 bp in P lac-wt spacer to create an HindIII site in making P lac did not change the transcription activity. Henceforth, P lac was used as a control in evaluating P [1][2][3][4][5][6] . The P [1][2][3][4][5][6] promoter showed Ͼ40-fold greater RNA synthesis compared with P lac in the absence of CRP.…”

Section: Resultsmentioning

confidence: 99%

“…Henceforth, P lac was used as a control in evaluating P [1][2][3][4][5][6] . The P [1][2][3][4][5][6] promoter showed Ͼ40-fold greater RNA synthesis compared with P lac in the absence of CRP. Whereas CRP (50 nM) stimulated P lac transcription 10-fold, it showed 2-fold further stimulation of P [1][2][3][4][5][6] .…”

Section: Resultsmentioning

confidence: 99%

“…To identify the critical base pairs in the Ϫ20 TTATGTT Ϫ13 sequence of P [1][2][3][4][5][6] that contribute to the high promoter activity, site-directed mutagenesis was used to make systematic single or multiple base pair changes in the spacer segment. Because of the presence of two additional T residues ( Ϫ22 TT Ϫ21 ) in the parental P lac , which is now 5Ј to the Ϫ20 TTTAT-GTT Ϫ13 sequence of P 1-6 , we scanned the entire segment from Ϫ22 to Ϫ13 (TTTTTATGTT).…”

Section: Resultsmentioning

confidence: 99%

“…The derivatives of P [1][2][3][4][5][6] were created by site-directed mutagenesis. The mutations were confirmed by DNA sequencing.…”

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A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

Liu

Tolstorukov

Zhurkin

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

108

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To determine whether the spacer region between the ؊35 and ؊10 elements plays any sequence-specific role, we randomized the GC-rich sequence ( ؊20 CCGGCTCG ؊13 ) within the spacer region of the cAMP-dependent lac promoter and selected an activatorindependent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the ؊15 sequence, which must be juxtaposed to the upstream end of the ؊10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a ؊35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the ؊18͞؊9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.A promoter is a stretch of DNA sequence that encodes information for RNA polymerase binding and initiation of transcription. Genetic and statistical analysis of promoters in Escherichia coli defined two kinds of E 70 RNA polymerase holoenzyme-dependent promoters (1). Both kinds require a 6-bp Ϫ10 sequence (consensus 5Ј-TATAAT-3Ј) located Ϸ7 bp 5Ј to the transcription start site. Functionally, the Ϫ10 element participates in RNA polymerase binding by interacting with the region 2.3-2.4 of 70 (2-10) and is part of an Ϸ15-nt putative single-stranded region in the open complex (4,8,(11)(12)(13)(14)(15). The first kind of promoters (Ϫ35 promoters) is more common and characterized by the presence of the Ϫ10 element as well as a 6-bp (consensus sequence 5Ј-TTGACA-3Ј) in the Ϫ35 position (16). The Ϫ35 element also helps RNA polymerase binding through interaction with region 4.2 of 70 (2,5,17). It is believed that the spacer region between Ϫ35 and Ϫ10 (optimal length 17 bp) does not have any specific sequence requirement and simply facilitates the spatial alignment of the Ϫ10 and Ϫ35 elements in binding to the 2.4 and 4.2 regions of the factor (7, 17, 18). The second kind, called ''extended Ϫ10'' promoters, contains an extra 2-bp 5Ј-TG-3Ј sequence located 1 bp 5Ј to the Ϫ10 element, (consensus sequence Ϫ15 TGNTATAAT Ϫ7 ) (19). The DNA sequence immediately upstream of an extended Ϫ10 element may enhance the activity of the extended Ϫ10 promoter (20, 21). The TG element also binds to RNA polymerase by contacting the 3.0 (formerly 2.5) segment of 70 (22). The Ϫ35 promoters may be improved by the presence of the extended Ϫ10 sequence and vice versa.Many naturally occurring promoters are more or less inefficient because of the presence of non-consensus sequence elements or suboptimal spacer lengths and are resurrected by extra regulatory factors (23). The extra factor may be a DNA sequence, e.g., an UP element (24-26). The UP element, located imme...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The derivatives of P [1][2][3][4][5][6] were created by site-directed mutagenesis. The mutations were confirmed by DNA sequencing.…”

mentioning

confidence: 99%

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A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

Liu

Tolstorukov

Zhurkin

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

108

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“…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).…”

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confidence: 99%

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

2000

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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...

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Structural and functional properties of aBacillus subtilis temperature-sensitive ?A factor

Wen¹,

Liao²,

Liou³

et al. 2000

Proteins

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Bacillus subtilis DB1005 is a temperature‐sensitive (Ts) sigA mutant containing double‐amino‐acid substitutions (I198A and I202A) on the hydrophobic face of the promoter −10 binding helix of σA factor. We have analyzed the structural and functional properties of this mutant σA factor both in vivo and in vitro. Our data revealed that the Ts σA factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts σA resulted in a very low core‐binding activity of the Ts σA factor and a markedly reduced σA‐RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts σA is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1‐anilinonaphthalene‐8‐sulfonate–binding analyses revealed that the hydrophobic face of the promoter −10 binding helix and also the hydrophobic core region of the Ts σA factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts σA and allows the formation of multimeric Ts σA. Our results also indicate that Ile‐198 and Ile‐202 on the hydrophobic face of the promoter −10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of σA factor. Proteins 2000;40:613–622. © 2000 Wiley‐Liss, Inc.

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Changes in conserved region 2 of Escherichia coli σ70 affecting promoter recognition

Cited by 185 publications

References 35 publications

A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

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

Structural and functional properties of aBacillus subtilis temperature-sensitive ?A factor

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Changes in conserved region 2 of Escherichia coli σ70 affecting promoter recognition

Cited by 185 publications

References 35 publications

A mutant spacer sequence between -35 and -10 elements makes the P lac promoter hyperactive and cAMP receptor protein-independent

A mutant spacer sequence between -35 and -10 elements makes the P lac promoter hyperactive and cAMP receptor protein-independent

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

Structural and functional properties of aBacillus subtilis temperature-sensitive ?A factor

Contact Info

Product

Resources

About

A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

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