Changes in the 17 bp spacer in the P R promoter of bacteriophage λ affect steps in open complex formation that precede DNA strand separation 1 1Edited by R. Ebright

McKane, Melissa; Gussin, Gary N.

doi:10.1006/jmbi.2000.3757

Cited by 14 publications

(18 citation statements)

References 42 publications

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“…The behavior of promoters recognized by E 70 and E 32 was similar in that both displayed a clear optimal distance, but the pronounced asymmetric pattern was atypical for E 70 promoters. In the few cases where asymmetry was observed with the latter, the promoter with the shorter spacer was the one with the lowest activity (22,32). The precipitous decline in promoter activity for the longer spacer length observed in Fig.…”

Section: Resultsmentioning

confidence: 72%

Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Wang

deHaseth

2003

J Bacteriol

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The Escherichia coli transcription factor sigma 32 binds to core RNA polymerase to form the holoenzyme responsible for transcription initiation at heat shock promoters, utilized upon exposure of the cell to higher temperatures. We have developed two ways to assay sigma 32-dependent RNA synthesis in E. coli. The plasmid-borne reporter gene for both is lacZ (␤-galactosidase), driven by the groE promoter. In one application, the cells are exposed to a temperature of 42°C in order to induce accumulation of endogenous sigma 32. The other involves isopropylthiogalactopyranoside (IPTG)-induced synthesis of sigma 32 at 30°C from a gene contained on a second plasmid. The latter employs DnaK ؊ cells, which additionally contained a second mutation, inactivating the endogenous sigma 32 gene (Bukau and Walker, EMBO J. 9:4027-4036, 1990). These assays were used to delineate the sequences CTTGA (؊37 to ؊33) and GNCCCCATNT (؊18 to ؊9) as important for sigma 32 promoter activity. At each of the specified base pairs, substitutions were found which reduced promoter activity by greater than 75%. Activity was also dependent upon the number of base pairs separating the two regions.RNA synthesis in prokaryotes is carried out by a multisubunit RNA polymerase commonly referred to as the core enzyme (E) (5). For promoter recognition, a sigma (initiation) factor is required. It interacts with the core polymerase to yield the holoenzyme (E), which is able to form an initiationcompetent complex at promoter sequences in a multistep process involving conformational changes in both the RNA polymerase and the promoter DNA (4, 5). In Escherichia coli, seven different species of the subunit have been identified, each directing transcription of a specific set of genes. The predominant sigma factor in E. coli is 70 , which imparts on RNA polymerase the ability to recognize promoters of housekeeping genes. The heat shock sigma factor of E. coli ( 32 ) is responsible for transcription of genes encoding proteins that promote survival of the cell at elevated temperatures (9,24,27,35).32 is a single polypeptide chain of 284 amino acids in length (19,35); it is a member of the 70 family of sigma factors, which share considerable homology in four distinct regions (21).The activity of 32 is increased at elevated growth temperatures and under conditions of nutrient starvation (7, 27, 34) as a result of multiple modes of regulation at the transcriptional, translational, and posttranslational levels (9,24,27,34 GroEL and GroES (9). From analysis of the sequences of 18 heat shock promoters, the consensus sequences CTTGAAA in the Ϫ35 region and CCCCATNT in the Ϫ10 region were derived (9). To date, no studies on the correlation of the consensus sequences and E 32 promoter function have been published. Thus, it was of interest to identify the sequences required for heat shock promoter function. We here describe a refinement of the consensus sequence as well as the development of two different assays for studying E 32 promoter activity in vivo which have enabled...

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Section: Resultsmentioning

confidence: 72%

Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Wang

deHaseth

2003

J Bacteriol

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“…In favorable cases, homogeneous populations of intermediates have been obtained by forming complexes at (or downshifting to) low temperature (cf. (5,11,(21)(22)(23)). For example, early complexes (I 1 or earlier species, presumably on the pathway to forming RP o ) have been characterized at binding equilibrium at low temperature, which slows or prevents their conversion to later intermediates (cf.…”

Section: (Mechanism I)mentioning

confidence: 99%

“…(11,13,21,24,25)), and at short times of reaction (9,10,22). Attempts to characterize I 2 at λP R by KMnO 4 footprinting after temperature-downshifts (11,23,26) appear to be complicated by the fact that the intrinsic KMnO 4 reactivity of the thymine bases in the open region of RP o may be strongly temperature dependent (27); M. Capp, unpublished). To date neither the amount nor the positions of DNA opening in I 2 have been unambiguously determined by downshift experiments.…”

Section: (Mechanism I)mentioning

confidence: 99%

Solute Probes of Conformational Changes in Open Complex (RP_o) Formation by Escherichia coli RNA Polymerase at the λP_R Promoter: Evidence for Unmasking of the Active Site in the Isomerization Step and for Large-Scale Coupled Folding in the Subsequent Conversion to RP_o

et al. 2006

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Transcription initiation is a multi-step process involving a series of requisite conformational changes in RNA polymerase (R) and promoter DNA (P) that create the open complex (RP o ). Here we use the small solutes urea and glycine betaine (GB) to probe the extent and type of surface area changes in the formation of RP o between Eσ 70 RNA polymerase and λP R promoter DNA. Effects of urea quantitatively reflect changes in amide surface and are particularly well suited to detect coupled protein folding events. GB provides a qualitative probe for the exposure or burial of anionic surface. Kinetics of formation and dissociation of RP o reveal strikingly large effects of the solutes on the final steps of RP o formation: urea dramatically increases the dissociation rate constant k d , whereas GB decreases the rate of dissociation. Formation of the first kinetically significant intermediate I 1 is disfavored in urea, and moderately favored by GB. GB slows the rate-determining step that converts I 1 to the second kinetically significant intermediate I 2 ; urea has no effect on this step. The most direct interpretation of these data is that recognition of promoter DNA in I 1 involves only limited conformational changes. Notably the data support the following hypotheses: 1) the negatively charged N-terminal domain of σ 70 remains bound in the "jaws" of polymerase in I 1 ; 2) the subsequent rate-determining isomerization step involves ejecting this domain from the jaws, thereby unmasking the active site; and 3) final conversion to RP o involves coupled folding of the mobile downstream clamp of polymerase.Kinetic studies of the process of open complex (RP o ) formation by E. coli RNA polymerase holoenzyme (R: catalytically competent core enzyme, α 2 ββ′ω bound to promoter DNArecognizing σ 70 subunit, also abbreviated Eσ 70 ) at the λP R promoter (P) have demonstrated that a minimum of three steps, involving two kinetically-significant intermediates (I 1 and I 2 ), are required to describe the mechanism (1-4): † This research was supported by NIH grant GM23467 to M. T. R. W. S. K. was supported by NIH Biotechnology Training grant 5 T32 GM08349.* To whom correspondence should be addressed. 433 Babcock Drive, Madison, WI 53706. Email: record@biochem.wisc.edu, rmsaecker@wisc.edu. ∥ These authors contributed equally to this work. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 27. Evidence for at least two kinetically significant intermediates has also been found for open complex formation at the lacUV5 (5), T7A1 (6-9) and λP RM up 1 promoters (10). Although the structures of these intermediates (I 1 , I 2 ) may be promoter-dependent (e.g. the extent to which DNA from -5 to +25 (where +1 is the start site) is protected by the "jaws" of RNAP in I 1 , cf. (9,11-13)), all proposed mechanisms for RP o formation at σ 70 promoters invoke a critical slow (rate-determining) isomerization step after formation of a first kinetically significant intermediate, I 1 . For λP R , this...

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“…The spacing between the Ϫ35 and Ϫ10 regions, at 17 nt, is the optimal spacing observed for C. perfringens (39) and E. coli (13) promoter sequences. This spacing appears to be a stringent constraint on promoter function because it is required for recognition by the RNA polymerase holoenzyme (5,14,30,33,47,51). Deviations from this spacing, as observed for the P3⌬A mutant with the reporter construct in this study, have a severe effect on expression.…”

Section: Discussionmentioning

confidence: 69%

Transcriptional Analysis of the tet (P) Operon from Clostridium perfringens

et al. 2001

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The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon. Deletion of P3 or alteration of the spacing between the ؊35 and ؊10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the tetA(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.The Tet P determinant from the 47-kb conjugative R-plasmid pCW3 from Clostridium perfringens is unique among tetracycline resistance determinants in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms. The tetA(P) gene is 1,260 bp in length and encodes a 46-kDa protein, TetA(P), which is responsible for the active efflux of tetracycline from the cell (44). The TetA(P) protein is predicted to contain 12 transmembrane domains but is atypical because it does not have the typical structure or conserved motifs that are common to the other classes of tetracycline efflux proteins (7, 21). The tetB(P) gene, which overlaps the tetA(P) gene by 17 nucleotides (nt), is 1,956 bp in length and encodes a putative 72.6-kDa protein. The TetB(P) protein has significant amino acid sequence identity (37 to 39%) to Tet(M)-like cytoplasmic ribosomal protection proteins (44).The tet(P) genes are the most widely distributed tetracycline resistance genes in C. perfringens, being found in both conjugative and nonconjugative tetracycline-resistant strains from diverse geographical locations and environmental sources (1, 24). Conjugative transfer of tetracycline resistance is invariably associated with plasmids that are either identical to or closely related to pCW3 (2,3,41). In these conjugative isolates, resistance is inducible. Inducible resistance is also observed when pCW3 is introduced into derivatives of strains CW234 and CW362, whereas in a strain 13 background tetracycline resistance is constitutively expressed, suggesting that induction requires an as-yet-unidentified host-encoded factor (19,38). Resistance is also constitutively expressed in nonconjugative isolates.Analysis of the approximately 1 kb of sequence data that are available upstream of tetA(P) (44) has revealed that this region is AT rich, having an overall GϩC content of 22%, which is similar to the normal 24 to 27% GϩC co...

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Changes in the 17 bp spacer in the P R promoter of bacteriophage λ affect steps in open complex formation that precede DNA strand separation 1 1Edited by R. Ebright

Cited by 14 publications

References 42 publications

Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Solute Probes of Conformational Changes in Open Complex (RP_o) Formation by Escherichia coli RNA Polymerase at the λP_R Promoter: Evidence for Unmasking of the Active Site in the Isomerization Step and for Large-Scale Coupled Folding in the Subsequent Conversion to RP_o

Transcriptional Analysis of the tet (P) Operon from Clostridium perfringens

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Changes in the 17 bp spacer in the P R promoter of bacteriophage λ affect steps in open complex formation that precede DNA strand separation 1 1Edited by R. Ebright

Cited by 14 publications

References 42 publications

Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Solute Probes of Conformational Changes in Open Complex (RPo) Formation by Escherichia coli RNA Polymerase at the λPR Promoter: Evidence for Unmasking of the Active Site in the Isomerization Step and for Large-Scale Coupled Folding in the Subsequent Conversion to RPo

Transcriptional Analysis of the tet (P) Operon from Clostridium perfringens

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Solute Probes of Conformational Changes in Open Complex (RP_o) Formation by Escherichia coli RNA Polymerase at the λP_R Promoter: Evidence for Unmasking of the Active Site in the Isomerization Step and for Large-Scale Coupled Folding in the Subsequent Conversion to RP_o