scholarly journals Transcription from the P3 Promoter of the Bacillus subtilis spx Gene Is Induced in Response to Disulfide Stress

2006 ◽  
Vol 189 (5) ◽  
pp. 1727-1735 ◽  
Author(s):  
Montira Leelakriangsak ◽  
Peter Zuber
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Intergenic Region ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Promoter Sequence ◽  
Primer Extension ◽  
Control Element ◽  
Start Site

ABSTRACT The spx gene of Bacillus subtilis encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with the yjbC gene. The spx gene DNA complements an spx null mutation with respect to disulfide stress resistance, suggesting that spx is transcribed from a promoter located in the intergenic region of yjbC and spx. Transcription of the yjbC-spx operon has been reported to be driven by four promoters, three (P1, P2, and PB) residing upstream of yjbC and one (PM) located in the intergenic region between yjbC and spx. Primer extension analysis uncovered a second intergenic promoter, P3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P3 is utilized by the σA form of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P3 did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position −40 of the P3 promoter start site. Deletion mutants with endpoints 3′ to the P3 transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P3 promoter sequence. Point mutations characterized by bgaB fusion expression and primer extension analyses uncovered evidence for a second cis-acting site in the P3 promoter sequence itself. The data indicate that spx transcription is under negative transcriptional control that is reversed when disulfide stress is encountered.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Dual Negative Control of spx Transcription Initiation from the P3 Promoter by Repressors PerR and YodB in Bacillus subtilis

2006 ◽  
Vol 189 (5) ◽  
pp. 1736-1744 ◽  
Author(s):  
Montira Leelakriangsak ◽  
Kazuo Kobayashi ◽  
Peter Zuber
Keyword(s):  
Oxidative Stress ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Point Mutations ◽  
Negative Control ◽  
Response To Treatment ◽  
Additive Increase

ABSTRACT The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P3 promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P3 promoter. Expression of an spx-bgaB fusion carrying the P3 promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P3 transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P3 promoter DNA was used as a template. Point mutations at positions within the P3 promoter relieved YodB-dependent repression, while a point mutation at position +24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P3 −10 and −35 regions, while PerR binds to a region downstream of the P3 transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis

1986 ◽  
Vol 6 (1) ◽  
pp. 227-235 ◽  
Author(s):  
M M Haltiner ◽  
S T Smale ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Upstream Region ◽  
Control Element ◽  
Rrna Gene ◽  
Start Site ◽  
Core Element ◽  
The Core ◽  
Polymerase I

A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.

scholarly journals The Length of Promoter Sequence Affects The De Novo Initiation By T7 RNA Polymerase in Vitro: New Insights Into The Evolution of Promoters For Single Subunit RNA Polymerases

2019 ◽  
Author(s):  
Ramesh Padmanabhan ◽  
Dennis Miller
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
De Novo ◽  
Promoter Sequence ◽  
Rna Polymerases ◽  
T7 Rna Polymerase ◽  
Promoter Sequences ◽  
Promoter Recognition ◽  
Single Subunit

1.1AbstractRNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate de novo transcription. Promoter recognition requires the presence of specific DNA binding domains in the polymerase. The structure and mechanistic aspects of transcription by the bacteriophage T7 RNA polymerase (T7 RNAP) are well characterized. This single subunit RNAP belongs to the family of RNAPs which also includes the T3, SP6 and mitochondrial RNAPs. High specificity for its promoter, the requirement of no additional transcription factors, and high fidelity of initiation from a specific site in the promoter makes it the polymerase of choice to study the mechanistic aspects of transcription. The structure and function of the catalytic domains of this family of polymerases are highly conserved suggesting a common mechanism underlying transcription. Although the two groups of single subunit RNAPs, mitochondrial and bacteriophage, have remarkable structural conservation, they recognize quite dissimilar promoters. Specifically, the bacteriophage promoters recognize a 23 nucleotide promoter extending from −17 to + 6 nucleotides relative to the site of transcription initiation, while the well characterized promoter recognized by the yeast mitochondrial RNAP is nine nucleotides in length extending from −8 to +1 relative to the site of transcription initiation. Promoters recognized by the bacteriophage RNAPs are also well characterized with distinct functional domains involved in promoter recognition and transcription initiation. Thorough mutational studies have been conducted by altering individual base-pairs within these domains. Here we describe experiments to determine whether the prototype bacteriophage RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. When a complete or almost complete (20 to 16 nucleotide) double stranded T7 RNAP promoter sequence is present, small RNA’s are produced through template-independent and promoter-dependent stuttering corresponding to abortive initiation, and this effect was lost with a scrambled promoter sequence. When partial double stranded promoter sequences (10 to 12 nucleotides) are supplied, template dependent de novo initiation of RNA occurs at a site different from the canonical +1-initiation site. The site of transcription initiation is determined by a recessed 3’ end based paired to the template strand of DNA rather than relative to the partial promoter sequence. Understanding the mechanism underlying this observation helps us to understand the role of the elements in the T7 promoter, and provides insights into the promoter evolution of the single-subunit RNAPs.

Transcription factor TFIIIB and transcription by RNA polymerase III

2006 ◽  
Vol 34 (6) ◽  
pp. 1082-1087 ◽  
Author(s):  
G.A. Kassavetis ◽  
E.P. Geiduschek
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Rna Polymerase Iii ◽  
Initiation Factor ◽  
Regulation Of Transcription ◽  
Start Site ◽  
High Mobility Group Protein ◽  
Polymerase Iii ◽  
Pol Iii

pol (RNA polymerase) III is charged with the task of transcribing nuclear genes encoding diverse small structural and catalytic RNAs. We present a brief review of the current understanding of several aspects of the pol III transcription apparatus. The focus is on yeast and, more specifically, on Saccharomyces cerevisiae; preponderant attention is given to the TFs (transcription initiation factors) and especially to TFIIIB, which is the core pol III initiation factor by virtue of its role in recruiting pol III to the transcriptional start site and its essential roles in forming the transcription-ready open promoter complex. Certain relatively recent developments are also selected for brief comment: (i) the genome-wide analysis of occupancy of pol III-transcribed genes (and other loci) by the transcription apparatus and the location of pol III transcription in the cell; (ii) progress toward a mechanistic and molecular understanding of the regulation of transcription by pol III in yeast; and (iii) recent experiments identifying a high mobility group protein as a fidelity factor that assures selection of the precise transcriptional start site at certain pol III promoters.

scholarly journals Functional Interaction between TFIIB and the Rpb2 Subunit of RNA Polymerase II: Implications for the Mechanism of Transcription Initiation

2004 ◽  
Vol 24 (9) ◽  
pp. 3983-3991 ◽  
Author(s):  
Bo-Shiun Chen ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Site Selection ◽  
Transcription Initiation ◽  
Growth Defect ◽  
Nucleoside Triphosphate ◽  
Start Site ◽  
Functional Interaction ◽  
Rnap Ii

ABSTRACT The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 → cysteine (R78C) replacement in the “B-finger” domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm−). Suppression of the R78C Csm− phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 → serine (G369S) replacement, located in the “lobe” domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 “lobe-jaw” region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIB complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.

scholarly journals Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

1986 ◽  
Vol 6 (1) ◽  
pp. 227-235 ◽  
Author(s):  
M M Haltiner ◽  
S T Smale ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Upstream Region ◽  
Control Element ◽  
Rrna Gene ◽  
Start Site ◽  
Core Element ◽  
The Core ◽  
Polymerase I

A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.

scholarly journals Properties of Bacillus subtilis σA Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

2004 ◽  
Vol 186 (8) ◽  
pp. 2366-2375 ◽  
Author(s):  
Hsin-Hsien Hsu ◽  
Wei-Cheng Huang ◽  
Jia-Perng Chen ◽  
Liang-Yin Huang ◽  
Chai-Fong Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Activity ◽  
Transcription Activity ◽  
N Terminus ◽  
Σ Factor

ABSTRACT σ factors in the σ70 family can be classified into the primary and alternative σ factors according to their physiological functions and amino acid sequence similarities. The primary σ factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative σ factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis σA, which belongs to a subgroup of the primary σ factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of σA, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated σA-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of σA in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the σA-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated σA and greatly reduced the transcription activity of the truncated σA-RNA polymerase by lowering the efficiency of transcription initiation after core binding of σA. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length σA in RNA polymerase.

scholarly journals Mutational Analysis of Yeast TFIIB: A Functional Relationship Between Ssu72 and Sub1/Tsp1 Defined by Allele-Specific Interactions With TFIIB

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 643-652
Author(s):  
Wei-Hua Wu ◽  
Inés Pinto ◽  
Bo-Shiun Chen ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Site Selection ◽  
Transcription Initiation ◽  
Mutational Analysis ◽  
Single Amino Acid ◽  
Structural Defects ◽  
Start Site ◽  
Allele Specific

Abstract TFIIB is an essential component of the RNA polymerase II core transcriptional machinery. Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro. In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo. A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two single amino acid replacements in TFIIB were defined and characterized. These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions. Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2μ) or enhancers (sub1Δ, ssu72-1) of sua7 mutations. We show that the interactions of ssu72-1 and sub1Δ with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II.

Sign in / Sign up

Export Citation Format

Share Document