scholarly journals Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Smarajit Mondal ◽  
Alexander V. Yakhnin ◽  
Paul Babitzke
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Untranslated Region ◽  
Modular Organization ◽  
Additional Time ◽  
Nascent Transcript ◽  
Content Type ◽  
Attenuation Mechanism ◽  
Transcription Attenuation ◽  
Rna Hairpin

ABSTRACT The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5′ untranslated region (5′ UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5′ UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5′ UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5′ UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding. IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5′ untranslated region (5′ UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5′ UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis trp operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.

scholarly journals Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Cierra A. Birch ◽  
Madison J. Davis ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Stress Induction ◽  
Content Type ◽  
Terminal Domain ◽  
Codon Substitution

ABSTRACT Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where “Y263C” indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

scholarly journals A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
David M. Rothstein ◽  
David Lazinski ◽  
Marcia S. Osburne ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Rna Synthesis ◽  
Bacterial Species ◽  
Ethanol Stress ◽  
Temperature Sensitive ◽  
Content Type ◽  
Promoter Recognition ◽  
Bacillis Subtilis

ABSTRACT Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.

scholarly journals rRNA (rrn) Operon-Engineered Bacillus subtilis as a Feasible Test Organism for Antibiotic Discovery

2013 ◽  
Vol 57 (4) ◽  
pp. 1948-1951 ◽  
Author(s):  
Yukinori Tanaka ◽  
Hideaki Nanamiya ◽  
Koichi Yano ◽  
Koji Kakugawa ◽  
Fujio Kawamura ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Test Organism ◽  
Rrna Operon ◽  
Content Type ◽  
Screening Experiments ◽  
Polymerase Inhibitors ◽  
Rifamycin Sv ◽  
Antibiotic Discovery

ABSTRACTBacillus subtiliscontains 10 rRNA (rrn) operons. We found that rRNA operon-engineeredB. subtilisstrain RIK543, with only therrnOoperon, is specifically hypersensitive to RNA polymerase inhibitors such as rifamycin SV and rifampin (80-fold and 20-fold, respectively). In pilot screening experiments, we found actinomycete isolates successfully at an incidence of 1.9% (18/945) that produced antibacterials that were detectable only with RIK543 as the test organism. Strain RIK543 may be a feasible test organism for the discovery of novel RNA polymerase inhibitors.

scholarly journals RNA Polymerase Trafficking in Bacillus subtilis Cells

2010 ◽  
Vol 192 (21) ◽  
pp. 5778-5787 ◽  
Author(s):  
Shu Ishikawa ◽  
Taku Oshima ◽  
Ken Kurokawa ◽  
Yoko Kusuya ◽  
Naotake Ogasawara
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Elongation Factor ◽  
Proximal Region ◽  
Elongation Complex ◽  
E Coli ◽  
Transcription Attenuation ◽  
Insight Into ◽  
Promoter Proximal Region

ABSTRACT To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the σA and β′ subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RΝΑP is evenly distributed from the promoter to the coding sequences. This finding suggests that, in general, B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex and proceeds without intragenic transcription attenuation. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which can be identified as transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior between E. coli and B. subtilis during initiation and elongation steps might result in distinct strategies for postinitiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis.

scholarly journals Molecular basis of TRAP-5'SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism

RNA ◽  
2008 ◽  
Vol 15 (1) ◽  
pp. 55-66 ◽  
Author(s):  
A. P. McGraw ◽  
A. Mokdad ◽  
F. Major ◽  
P. C. Bevilacqua ◽  
P. Babitzke
Keyword(s):  
Bacillus Subtilis ◽  
Molecular Basis ◽  
Attenuation Mechanism ◽  
Transcription Attenuation ◽  
Rna Interaction ◽  
Trp Operon

scholarly journals trp RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBAOperon

2000 ◽  
Vol 182 (7) ◽  
pp. 1819-1827 ◽  
Author(s):  
Hansen Du ◽  
Alexander V. Yakhnin ◽  
Subramanian Dharmaraj ◽  
Paul Babitzke
Keyword(s):  
Bacillus Subtilis ◽  
Rna Structure ◽  
Rna Binding ◽  
Structure Mapping ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Expression Studies ◽  
Attenuation Mechanism ◽  
Transcription Attenuation ◽  
Rna Interaction

ABSTRACT The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBAoperon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5′ end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5′ stem-loop increases the affinity of TRAP fortrp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5′ stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5′ stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE′-′lacZtranslational fusions demonstrate that TRAP-5′ stem-loop interaction is required for proper regulation of the trp operon. 3′ RNA boundary experiments indicate that the 5′ structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5′ stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

scholarly journals NusG-Dependent RNA Polymerase Pausing and Tylosin-Dependent Ribosome Stalling Are Required for Tylosin Resistance by Inducing 23S rRNA Methylation in Bacillus subtilis

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Helen Yakhnin ◽  
Alexander V. Yakhnin ◽  
Brandon L. Mouery ◽  
Zachary F. Mandell ◽  
Catherine Karbasiafshar ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Macrolide Antibiotic ◽  
Transcription Termination ◽  
Leader Peptide ◽  
23S Rrna ◽  
Content Type ◽  
Ribosome Stalling ◽  
Exit Tunnel ◽  
Polymerase Pausing

ABSTRACT Macrolide antibiotics bind to 23S rRNA within the peptide exit tunnel of the ribosome, causing the translating ribosome to stall when an appropriately positioned macrolide arrest motif is encountered in the nascent polypeptide. Tylosin is a macrolide antibiotic produced by Streptomyces fradiae. Resistance to tylosin in S. fradiae is conferred by methylation of 23S rRNA by TlrD and RlmAII. Here, we demonstrate that yxjB encodes RlmAII in Bacillus subtilis and that YxjB-specific methylation of 23S rRNA in the peptide exit tunnel confers tylosin resistance. Growth in the presence of subinhibitory concentrations of tylosin results in increased rRNA methylation and increased resistance. In the absence of tylosin, yxjB expression is repressed by transcription attenuation and translation attenuation mechanisms. Tylosin-dependent induction of yxjB expression relieves these two repression mechanisms. Induction requires tylosin-dependent ribosome stalling at an RYR arrest motif at the C terminus of a leader peptide encoded upstream of yxjB. Furthermore, NusG-dependent RNA polymerase pausing between the leader peptide and yxjB coding sequences is essential for tylosin-dependent induction. Pausing synchronizes the position of RNA polymerase with ribosome position such that the stalled ribosome prevents transcription termination and formation of an RNA structure that sequesters the yxjB ribosome binding site. On the basis of our results, we are renaming yxjB as tlrB. IMPORTANCE Antibiotic resistance is a growing health concern. Resistance mechanisms have evolved that provide bacteria with a growth advantage in their natural habitat such as the soil. We determined that B. subtilis, a Gram-positive soil organism, has a mechanism of resistance to tylosin, a macrolide antibiotic commonly used in the meat industry. Tylosin induces expression of yxjB, which encodes an enzyme that methylates 23S rRNA. YxjB-dependent methylation of 23S rRNA confers tylosin resistance. NusG-dependent RNA polymerase pausing and tylosin-dependent ribosome stalling induce yxjB expression, and hence tylosin resistance, by preventing transcription termination upstream of the yxjB coding sequence and by preventing repression of yxjB translation.

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