Mutation changing the specificity of an RNA polymerase sigma factor

Zuber, Peter; Healy, J M; Carter, H.Luke; Cutting, Simon M.; Moran, Charles P.; Losick, Richard

doi:10.1016/0022-2836(89)90569-x

Cited by 178 publications

(172 citation statements)

References 37 publications

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“…We quantified 76-to 77-nt long run-off RNA made in vitro. The results of transcription with P lac-wt, P lac , and P [1][2][3][4][5][6] in the absence and presence of CRP are shown in Fig. 2.…”

Section: Resultsmentioning

confidence: 99%

“…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)(3)(4)(5)(6)(7)(8)(9)(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).…”

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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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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%

mentioning

confidence: 99%

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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“…In addition, transcription of spo0A itself is controlled in part by sigma-H (4,22,37), and the sigma-H-controlled promoter upstream of spo0A, Ps, is required for sporulation (29). Other sigma-H-controlled genes or promoters include spoIIA, spoVG, rpoDP3, rpoDP4, and ftsZP2 (3,11,24,36,40).…”

mentioning

confidence: 99%

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

Hicks

Grossman

1995

J Bacteriol

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spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The csh203 insertion mutation caused a 2-to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.

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“…Temporally regulated transcription from the 18-bp sapA promoter does not fit well with current models of promoter recognition by most prokaryotic sigma factors that invoke key contacts to sequences around Ϫ35 and Ϫ10 bp upstream (11,27,34). Therefore, as in eukaryotic TATA-less promoters, it is possible that a protein binding at or near the transcription start site mediates formation of a transcription complex.…”

Section: Discussionmentioning

confidence: 73%

The sapA promoter from Streptomyces coelicolor requires activation sites and initiator-like sequences but No -10 or -35 sequences

1995

J Bacteriol

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The Streptomyces coelicolor sapA gene encodes a spore coat protein. The sapA promoter is regulated developmentally, with maximal expression occurring in aerial hyphae at a late stage of colonial development. The DNA sequences upstream from the transcription start point do not appear to fall into a previously described promoter class. One (or more) putative activation site, required for full activity, is eliminated when 5 deletions extend to between ؊178 and ؊72 bases upstream from the transcription start point. In addition, a downstream activation site is destroyed by removing sequences between positions ؉40 and ؉120, relative to the transcription start point, in the absence of an intact upstream region. However, temporal regulation of transcription initiation over the course of the life cycle is maintained faithfully in the absence of these elements, even in the smallest 18-bp sapAp fragment containing sequences from positions ؊8 to ؉10. Site-specific mutations around the transcriptional start points shift the timing of sapA expression to an earlier stage in the developmental cycle. These results suggest that a novel mechanism may be involved in Streptomyces late gene expression.

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Mutation changing the specificity of an RNA polymerase sigma factor

Cited by 178 publications

References 37 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

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

The sapA promoter from Streptomyces coelicolor requires activation sites and initiator-like sequences but No -10 or -35 sequences

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Mutation changing the specificity of an RNA polymerase sigma factor

Cited by 178 publications

References 37 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

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

The sapA promoter from Streptomyces coelicolor requires activation sites and initiator-like sequences but No -10 or -35 sequences

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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