RESUMEN
Each peptide bond of a protein is generated at the peptidyl transferase center (PTC) of the ribosome and then moves through the exit tunnel, which accommodates ever-changing segments of ≈ 40 amino acids of newly translated polypeptide. A class of proteins, called ribosome arrest peptides, contains specific sequences of amino acids (arrest sequences) that interact with distinct components of the PTC-exit tunnel region of the ribosome and arrest their own translation continuation, often in a manner regulated by environmental cues. Thus, the ribosome that has translated an arrest sequence is inactivated for peptidyl transfer, translocation, or termination. The stalled ribosome then changes the configuration or localization of mRNA, resulting in specific biological outputs, including regulation of the target gene expression and downstream events of mRNA/polypeptide maturation or localization. Living organisms thus seem to have integrated potentially harmful arrest sequences into elaborate regulatory mechanisms to express genetic information in productive directions.
Asunto(s)
Péptidos/química , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Humanos , Péptidos/genética , Péptidos/metabolismo , Ribosomas/genéticaRESUMEN
Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.
RESUMEN
Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Biosíntesis de Proteínas , Ribosomas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Ribosomas/metabolismo , Dominios Proteicos , Microscopía por Crioelectrón , Unión Proteica , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Modelos Moleculares , Aminoacil-ARN de TransferenciaRESUMEN
The ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. We examined ribosome ubiquitination during zebrafish development by combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis. Our findings showed that the ubiquitination of ribosomal proteins dynamically changed as development proceeded. We also showed that during zebrafish development, the ribosome was ubiquitinated by Znf598, an E3 ubiquitin ligase that activates RQC. Ribosomal protein Rps10/eS10 was found to be a key ubiquitinated protein during development. Furthermore, we showed that Rps10/eS10 ubiquitination-site mutations reduced the overall ubiquitination pattern of the ribosome. These results demonstrate the complexity and dynamics of ribosome ubiquitination during zebrafish development.
Asunto(s)
Biosíntesis de Proteínas , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Ribosomas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ubiquitinación , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Magnesio/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Conformación Proteica , Estabilidad Proteica , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Ribosomas/química , Ribosomas/genética , Relación Estructura-ActividadRESUMEN
Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Genes Bacterianos , Bacterias Grampositivas , Antibacterianos/farmacología , Antibacterianos/química , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/genética , Nucleósidos/química , Nucleósidos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Genes Bacterianos/genética , Clostridium/efectos de los fármacos , Clostridium/genética , Microscopía por CrioelectrónRESUMEN
Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.
Asunto(s)
Antibacterianos , Bacillus subtilis , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Microbiana/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato/metabolismo , Factores R , Transcripción GenéticaRESUMEN
Regulatory nascent peptides participate in the regulation of cellular functions by the mechanisms involving regulated translation arrest. A class of them in bacteria, called monitoring substrates, feedback-regulates the expression of a specific component of protein localization machinery. Three monitoring substrates, SecM, MifM and VemP have previously been identified. Here, we attempt at identifying additional arrest peptides in bacteria. Our bioinformatic searches over more than 400 bacterial genomic sequences for proteins that have the common characteristic features shared by the known monitoring substrates and subsequent in vitro and in vivo characterization of the highlighted sequences allowed the identification of three arrest peptides termed ApcA, ApdA and ApdP. ApcA and ApdA homologs are conserved among a subset of actinobacteria, whereas ApdP has homologs in a subset of α-proteobacteria. We demonstrate that these arrest peptides, in their ribosome-tethered nascent states, inhibit peptidyl transfer. The elongation arrest occurs at a specific codon near the 3' end of the coding region, in a manner depending on the amino acid sequence of the nascent chain. Interestingly, the arrest sequences of ApcA, ApdA and ApdP share a sequence R-A-P-G/P that is essential for the elongation arrest.
Asunto(s)
Extensión de la Cadena Peptídica de Translación , Péptidos/química , Actinobacteria/genética , Alphaproteobacteria/genética , Codón , Biología Computacional , Mutación , Sistemas de Lectura Abierta , Péptidos/genética , Biosíntesis de Proteínas , RibosomasRESUMEN
Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.
Asunto(s)
Bacillus/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Arginina/metabolismo , Membrana Celular/química , Secuencia Conservada , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Bacillus subtilis MifM uses polypeptide-instructed ribosomal stalling to control translation of YidC2, a membrane protein biogenesis factor. In contrast to other stalling systems involving a single arrest point, our in vitro translation/toeprint experiments show that the B. subtilis ribosome stalls consecutively at multiple codons of MifM. This mode of elongation arrest depends on nascent chain residues at the middle of the ribosomal exit tunnel and a few (four for the maximum functionality) negative charges residing proximally to the arrest points. The latter element does not require exact amino acid sequence, and this feature may underlie the multisite stalling. The arrested nascent chains were not efficiently transferred to puromycin, suggesting that growing MifM nascent chains inhibit peptidyl transferase center after acquiring an acidic residue(s). Multisite stalling seems to provide a unique means for MifM to achieve a sufficient duration of ribosomal stalling required for the regulatory function.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas de Transporte de Membrana/biosíntesis , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/genética , Peptidil Transferasas/metabolismo , Biosíntesis de ProteínasRESUMEN
Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.
Asunto(s)
Biosíntesis de Proteínas , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Genes Bacterianos , Puromicina/farmacología , ARN de Transferencia/genéticaRESUMEN
The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Transporte de Membrana/química , Arginina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Escherichia coli/metabolismo , Etilmaleimida/química , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Transporte de Membrana/metabolismo , Estructura Terciaria de Proteína , Agua/químicaRESUMEN
SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine-Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles.
Asunto(s)
Proteínas Bacterianas/genética , Biosíntesis de Proteínas , Tolerancia a la Sal/genética , Vibrio/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Immunoblotting , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Transporte de Proteínas/genética , Fuerza Protón-Motriz/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Salinidad , Agua de Mar/microbiología , Homología de Secuencia de Aminoácido , Sodio/metabolismo , Vibrio/metabolismoRESUMEN
The YidC/Oxa1/Alb3 family proteins are involved in membrane protein biogenesis in bacteria, mitochondria, and chloroplasts. Recent studies show that YidC uses a channel-independent mechanism to insert a class of membrane proteins into the membrane. Bacillus subtilis has two YidC homologs, SpoIIIJ (YidC1) and YidC2 (YqjG); the former is expressed constitutively, while the latter is induced when the SpoIIIJ activity is compromised. MifM is a substrate of SpoIIIJ, and its failure in membrane insertion is accompanied by stable ribosome stalling on the mifM-yidC2 mRNA, which ultimately facilitates yidC2 translation. While mutational inactivation of SpoIIIJ has been known to induce yidC2 expression, here, we show that the level of this induction is lower than that observed when the membrane insertion signal of MifM is defective. Moreover, this partial induction of YidC2 translation is lowered further when YidC2 is overexpressed in trans. These results suggest that YidC2 is able to insert MifM into the membrane and to release its translation arrest. Thus, under SpoIIIJ-deficient conditions, YidC2 expression is subject to MifM-mediated autogenous feedback repression. Our results show that YidC2 uses a mechanism that is virtually identical to that used by SpoIIIJ; Arg75 of YidC2 in its intramembrane yet hydrophilic cavity is functionally indispensable and requires negatively charged residues of MifM as an insertion substrate. From these results, we conclude that MifM monitors the total activities of the SpoIIIJ and the YidC2 pathways to control the synthesis of YidC2 and to maintain the cellular capability of the YidC mode of membrane protein biogenesis.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Bacillus subtilis/genética , Proteínas Bacterianas/genéticaRESUMEN
Nascent chain-mediated translation arrest serves as a mechanism of gene regulation. A class of regulatory nascent polypeptides undergoes elongation arrest in manners controlled by the dynamic behavior of the growing chain; Escherichia coli SecM monitors the Sec protein export pathway and Bacillus subtilis MifM monitors the YidC membrane protein integration/folding pathway. We show that MifM and SecM interact with the ribosome in a species-specific manner to stall only the ribosome from the homologous species. Despite this specificity, MifM is not exclusively designed to monitor membrane protein integration because it can be converted into a secretion monitor by replacing the N-terminal transmembrane sequence with a secretion signal sequence. These results show that a regulatory nascent chain is composed of two modular elements, one devoted to elongation arrest and another devoted to subcellular targeting, and they imply that physical pulling force generated by the latter triggers release of the arrest executed by the former. The combinatorial nature may assure common occurrence of nascent chain-mediated regulation.
Asunto(s)
Bacillus subtilis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación de la Expresión Génica , Biosíntesis de Proteínas/genética , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Especificidad de la EspecieRESUMEN
A signal peptide (SP) is cleaved off from presecretory proteins by signal peptidase during or immediately after insertion into the membrane. In metazoan cells, the cleaved SP then receives proteolysis by signal peptide peptidase, an intramembrane-cleaving protease (I-CLiP). However, bacteria lack any signal peptide peptidase member I-CLiP, and little is known about the metabolic fate of bacterial SPs. Here we show that Escherichia coli RseP, an site-2 protease (S2P) family I-CLiP, introduces a cleavage into SPs after their signal peptidase-mediated liberation from preproteins. A Bacillus subtilis S2P protease, RasP, is also shown to be involved in SP cleavage. These results uncover a physiological role of bacterial S2P proteases and update the basic knowledge about the fate of signal peptides in bacterial cells.
Asunto(s)
Bacillus subtilis/enzimología , Endopeptidasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de la Membrana/metabolismo , Señales de Clasificación de Proteína , Catálisis , HidrólisisRESUMEN
During λ infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105-GFP chimera and real-time deconvolution fluorescence microscopy to show that the S105-GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an early-lysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105(ΔTMD1)-mcherryfp, in the product of which the S105 sequence deleted for the first transmembrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105-GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105-GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis.
Asunto(s)
Regulación Viral de la Expresión Génica , Proteínas Virales/fisiología , Alelos , Secuencia de Aminoácidos , Apoptosis , Genes Dominantes , Proteínas Fluorescentes Verdes/metabolismo , Luz , Microdominios de Membrana , Microscopía Fluorescente/métodos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas Virales/genéticaRESUMEN
Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.
Asunto(s)
Proteínas de Escherichia coli , Biosíntesis de Proteínas , Filogenia , Péptidos/química , Ribosomas/genética , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.
Asunto(s)
Peptidil Transferasas , Peptidil Transferasas/metabolismo , Antibacterianos/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Péptidos/metabolismo , ARN de Transferencia/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.