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.

scholarly journals Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling

2020 ◽  
Author(s):  
Tatsuma Shoji ◽  
Akiko Takaya ◽  
Yoko Kusuya ◽  
Hiroki Takahashi ◽  
Hiroto Kawashima
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ribosome Profiling ◽  
23S Rrna ◽  
Ribosome Stalling ◽  
70S Ribosome ◽  
Exit Tunnel ◽  
Species Specific ◽  
Peptidyltransferase Center ◽  
First Time

2.Abstract(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant.(2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling.(3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

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 The expression of antibiotic resistance methyltransferase correlates with mRNA stability independently of ribosome stalling

2016 ◽  
pp. AAC.01806-16 ◽  
Author(s):  
Ekaterina Dzyubak ◽  
Mee-Ngan F. Yap
Keyword(s):  
Leader Peptide ◽  
23S Rrna ◽  
Cross Resistance ◽  
Nonsense Mutations ◽  
Stem Loop ◽  
Translational Initiation ◽  
Ribosome Stalling ◽  
Mrna Structure ◽  
Bacterial Ribosome ◽  
A Site

Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression ofermis activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the co-transcribederm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes theermShine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here we show that nonsense mutations in theStaphylococcus aureusErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence and presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of theermBL-ermBtranscript, and macrolide appears to mitigate mRNA cleavage at a site immediately downstream of theermBLSD sequence. The stabilizing effect of antibiotics on mRNA is not limited toermBL-ermB; cationic antibiotics representing a ribosome stalling inducer and a non-inducer increase the half-life of specific transcripts. These data unveil a new layer ofermBregulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

scholarly journals Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

2021 ◽  
Author(s):  
Corentin R. Fostier ◽  
Farès Ousalem ◽  
Elodie Carmen Leroy ◽  
Saravuth Ngo ◽  
Heddy Soufari ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Macrolide Resistance ◽  
Leader Peptide ◽  
23S Rrna ◽  
Human Pathogens ◽  
F Protein ◽  
Release Factors ◽  
Translation Factors ◽  
Exit Tunnel ◽  
Mechanistic Basis

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the activity of the streptococcal ARE ABC-F protein MsrD on the ribosome and its regulation in response to macrolide exposure. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into a conserved crevice of the ribosomal exit tunnel, which remained thus far undocumented, concomitantly with 23S rRNA rearrangements that preclude proper accommodation of release factors and inhibits termination. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD powered by its two functionally asymmetric ATPase sites, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

scholarly journals An antibiotic-sensing leader peptide regulates translation and premature Rho-dependent transcription termination of thetopAIgene inEscherichia coli

2019 ◽  
Author(s):  
Gabriele Baniulyte ◽  
Joseph T. Wade
Keyword(s):  
Conformational Changes ◽  
Rna Structure ◽  
Transcription Termination ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Translational Repression ◽  
Leader Peptide ◽  
Rna Structures ◽  
Ribosome Stalling ◽  
Specific Manner

AbstractLong 5′ UTRs in bacteria often contain regulatory elements that modulate expression of the downstream gene in response to environmental stimuli. In most examples of such regulation, the mechanism involves switching between alternative 5′ UTR RNA structures that impact transcription, stability, or translation of the mRNA. Here, we show that transcription of theEscherichia coli topAIgene is prematurely terminated by the termination factor Rho under standard laboratory growth conditions, and that this occurs as a result of translational repression. Regulation oftopAItranslation is controlled by a sensory ORF,toiL, located within thetopAI5′ UTR. We show that ribosomes translatingtoiLstall in a sequence-specific manner in the presence of specific ribosome-targeting antibiotics. Ribosome stalling attoiLinduces conformational changes in the RNA structure of thetopAI5′ UTR, unmasking thetopAIribosome-binding site, thereby relieving translational repression and preventing premature transcription termination. Thus,toiLacts as a sensor of translation stress, leading to regulation oftopAIat both the translational and transcriptional levels.

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.

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