Reduced capacity of alternative σs to melt promoters ensures stringent promoter recognition

Koo, Bon Chul; Rhodius, Virgil A.; Nonaka, Gen; deHaseth, Pieter L.; Gross, Carol A.

doi:10.1101/gad.1843709

Cited by 39 publications

(52 citation statements)

References 68 publications

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“…Further, it was demonstrated that RpoH from the alphaproteobacterium Caulobacter crescentus recognizes E. coli promoters that lack the extended Ϫ10 motif (29). It has been postulated that by increasing promoter stringency, the extended Ϫ10 motif allows for an efficient response to a restricted set of promoters and that lack of an extended motif may allow alphaproteobacterial RpoH to transcribe larger sets of genes involved in a variety of processes (30). This premise is supported by previous work showing that, in addition to heat shock, S. meliloti rpoH1 is required for response to acid shock and survival in root nodules (10,40,47) and by our current study defining a large set of rpoH-dependent genes.…”

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confidence: 99%

Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium

et al. 2012

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ABSTRACTSinorhizobium melilotican live as a soil saprophyte and can engage in a nitrogen-fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma (σ) factors interacting with core RNA polymerase. TheS. melilotigenome encodes 14 of these alternative σ factors, including two putative RpoH (“heat shock”) σ factors. We used custom Affymetrix symbiosis chips to characterize the global transcriptional response ofS. melilotirpoH1,rpoH2, andrpoH1 rpoH2mutants during heat shock and stationary-phase growth. Under these conditions, expression of over 300 genes is dependent onrpoH1andrpoH2. We mapped transcript start sites of 69rpoH-dependent genes using 5′ RACE (5′ rapid amplification of cDNA ends), which allowed us to determine putative RpoH1-dependent, RpoH2-dependent, and dual-promoter (RpoH1- and RpoH2-dependent) consensus sequences that were each used to search the genome for other potential direct targets of RpoH. The inferredS. melilotiRpoH promoter consensus sequences share features ofEscherichia coliRpoH promoters but lack extended −10 motifs.

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Section: Discussionmentioning

confidence: 99%

Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium

et al. 2012

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“…However, the evidence is, in general, weak (Discussion) (9,22,23). Effective experimental tests have been complicated by (i) the general inability of bulk experiments to directly measure promoter binding uncoupled from subsequent conformational changes (2, 24) and (ii) the weak sequence specificity of DNA binding (25,26).In a previous study (7), we used colocalization single-molecule spectroscopy (CoSMoS) to directly observe and measure the initial rate of binding of σ 54 RNAP to a cognate promoter uncoupled from subsequent conformational isomerization of the polymerasepromoter complex. We also showed that the initial RNAPpromoter complex that we observe is on the pathway to transcription initiation.…”

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RNA polymerase approaches its promoter without long-range sliding along DNA

Friedman

Mumm

Gelles

2013

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

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Sequence-specific DNA binding proteins must quickly bind target sequences, despite the enormously larger amount of nontarget DNA present in cells. RNA polymerases (or associated general transcription factors) are hypothesized to reach promoter sequences by facilitated diffusion (FD). In FD, a protein first binds to nontarget DNA and then reaches the target by a 1D sliding search. We tested whether Escherichia coli σ 54 RNA polymerase reaches a promoter by FD using the colocalization single-molecule spectroscopy (CoSMoS) multiwavelength fluorescence microscopy technique. Experiments directly compared the rates of initial polymerase binding to and dissociation from promoter and nonpromoter DNAs measured in the same sample under identical conditions. Binding to a nonpromoter DNA was much slower than binding to a promoter-containing DNA of the same length, indicating that the detected nonspecific binding events are not on the pathway to promoter binding. Truncating one of the DNA segments flanking the promoter to a length as short as 7 bp or lengthening it to ∼3,000 bp did not alter the promoter-specific binding rate. These results exclude FD over distances corresponding to the length of the promoter or longer from playing any significant role in accelerating promoter search. Instead, the data support a direct binding mechanism, in which σ 54 RNA polymerase reaches the local vicinity of promoters by 3D diffusion through solution, and suggest that binding may be accelerated by atypical structural or dynamic features of promoter DNA. Direct binding explains how polymerase can quickly reach a promoter, despite occupancy of promoter-flanking DNA by bound proteins that would impede FD.1D diffusion | total internal reflection fluorescence | transcription initiation T he binding of a multisubunit RNA polymerase (RNAP) or general transcription factors to a specialized transcription promoter DNA sequence is an essential step in initiating DNA transcription in all organisms (1, 2). Control of this promoter binding step is a key mechanism by which gene expression is regulated (3). To understand how this regulation occurs, it is necessary to understand the process by which the transcription machinery efficiently finds and binds to target sequences embedded in chromosomal DNA, where targets are outnumbered by orders of magnitude excess nontarget DNA.Bacterial transcription is a convenient model system for studying promoter search, because the process can be efficiently reconstituted in vitro from purified components and bacterial RNAP holoenzymes recognize and bind directly to specific promoter DNA sequence motifs (4, 5). For the well-studied Escherichia coli RNAP, the rate at which the enzyme finds its cognate promoters can approach or even possibly exceed limits calculated for simple 3D diffusion of the protein up to the target sequence followed by direct promoter binding (6-8). Although there are uncertainties in calculating the magnitude of this limit because of uncertain contributions from orientation constraints and electr...

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“…The GCCG motif is a composite recognition element: the GC is recognized by R91 from region 3 of E. coli 28 (the D ortholog), whereas the CG is recognized by R84 and D81 from region 2.4 (14). The corresponding residues in R91 recognizes a G residue at either of the first two positions of the extended Ϫ10 element (15), and D Ap R104 is therefore predicted to contact G on the template strand at position Ϫ14 (Fig. 2).…”

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DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

et al. 2011

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D proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the ؊10 element, and this motif is known to be recognized by region 2.4 and critical for promoter activity.In the course of efforts to define gene expression determinants from the thermophilic bacterium Aeribacillus pallidus AC6 (2, 18), we identified flagellin (Hag) as being among the most highly expressed proteins in this strain. To determine the basis for high-level Hag expression, we isolated and sequenced the hag gene and identified and expressed the protein required for its expression in The resulting peptide sequences displayed high similarity to B. subtilis flagellin (NAQDGISLIQTAEGALTETHAILQR had 96% identity with amino acids [aa] 65 to 89 and LEHTINNL GTSAENLTAAESR had 85% identity with aa 242 to 262). Two degenerate primers (FlaF1 and FlaR1) were used to amplify the flagellin gene (hag) from A. pallidus AC6 chromosomal DNA. An ϳ450-bp product was cloned into pGEM-T Easy (Promega) for DNA sequencing. The remainder of hag and its upstream region were obtained by inverse PCR. The 828-bp hag gene encodes a 275-amino-acid (29.7-kDa) protein having 63% identity with B. subtilis Hag and is preceded by a typical D promoter. To identify sigD, two degenerate primers were used to amplify a PCR fragment which was cloned into the pGEM-T Easy vector system and sequenced. The flanking portions of sigD were obtained by inverse PCR, and the gene was sequenced. The 771-bp sigD gene encodes a 256-amino-acid (28.7-kDa) protein having 67% overall identity with D Bs , with the highest levels of similarity concentrated in conserved regions 2 and 4, known to mediate promoter recognition.A. pallidus AC6 flagellin is expressed from a D -dependent promoter. Transcription of hag initiates from a canonical Ddependent promoter at a G residue 79 bp upstream of the start codon (Fig. 1). Analysis of hag::lacZ fusions integrated into B. subtilis CU1065 and HB4035 (sigD::kan) indicated that activity was D dependent and highest at late logarithmic phase, as previously reported for B. subtilis (19). Optimal promoter activity required an AT-rich region just upstream of the Ϫ35 element (Table 1), which has similarity with the upstream promoter (UP) element previously described for B. subtilis hag (6). Sequence inspection suggests that high-level Hag expression may also benefit from a strong ribosome-binding site and stabilization of the mRNA by a 5Ј hairpin sequence (24). Purification D of A. pallidus and B. subtilis and reconstitution of D RNAP. D proteins from A. pallidus AC6 and B. subtilis were expressed under T7 RNA polymerase (RNAP) control in Escherichia coli BL21(DE3)/pLysS (Novagen) by using pECG1 and pECB1 (Table 2). For purification, inclusion bodies were solubilized with Sarkosyl (1), refolded, and purified using DEAE-Sepharose and heparin-Sepharose chromatography as described previously (9). B. subtilis core RNAP was purified...

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Reduced capacity of alternative σs to melt promoters ensures stringent promoter recognition

Cited by 39 publications

References 68 publications

Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium

Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium

RNA polymerase approaches its promoter without long-range sliding along DNA

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

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Reduced capacity of alternative σs to melt promoters ensures stringent promoter recognition

Cited by 39 publications

References 68 publications

Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium

Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium

RNA polymerase approaches its promoter without long-range sliding along DNA

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ D Proteins

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Product

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DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins