Proteome-wide determinants of co-translational chaperone binding in bacteria
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ABSTRACT:
PROVIDER: PRJNA1238275 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32773106.fastq.gz | Fastqsanger.gz | |||
| SRR32773107.fastq.gz | Fastqsanger.gz | |||
| SRR32773108.fastq.gz | Fastqsanger.gz | |||
| SRR32773109.fastq.gz | Fastqsanger.gz | |||
| SRR32773110.fastq.gz | Fastqsanger.gz |
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Project description:Chaperones are essential to the co-translational folding of most proteins. However, the principles of co-translational chaperone interaction throughout the proteome are poorly understood, as current methods are restricted to few substrates and cannot capture nascent protein folding or chaperone binding sites, precluding a comprehensive understanding of productive and erroneous protein biosynthesis. Here, by integrating genome-wide selective ribosome profiling, single-molecule tools, and computational predictions using AlphaFold we show that the binding of the main E. coli chaperones involved in co-translational folding, Trigger Factor (TF) and DnaK correlates with “unsatisfied residues” exposed on nascent partial folds – residues that have begun to form tertiary structure but cannot yet form all native contacts due to ongoing translation. This general principle allows us to predict their co-translational binding across the proteome based on sequence only, which we verify experimentally. The results show that TF and DnaK stably bind partially folded rather than unfolded conformers. They also indicate a synergistic action of TF guiding intra-domain folding and DnaK preventing premature inter-domain contacts, and reveal robustness in the larger chaperone network (TF, DnaK, GroEL). Given the complexity of translation, folding, and chaperone functions, our predictions based on general chaperone binding rules indicate an unexpected underlying simplicity.
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Project description:Molecular chaperones assist in protein folding by interacting with nascent polypeptide chains (NCs) during translation, but whether the ribosome can sense chaperone defects and abort translation of misfolding NCs has not been explored. Here we used quantitative proteomics in E. coli to investigate the ribosome-associated chaperone network and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL and ClpB contribute increasingly, when DnaK is deficient. Surprisingly, misfolding of NCs due to defects in co-translational chaperone function or amino acid analog incorporation, results in recruitment of the non-canonical release factor RF3 to the ribosome. RF3 then cooperates with RF2 in mediating premature chain termination of a subset of abundant NCs, allowing their efficient degradation. This function of RF3 reduces the accumulation of misfolded proteins. It is critical for proteostasis maintenance and cell growth under conditions of limited chaperone availability.
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Project description:Molecular chaperones assist in protein folding by interacting with nascent polypeptide chains (NCs) during translation, but whether the ribosome can sense chaperone defects and abort translation of misfolding NCs has not been explored. Here we used quantitative proteomics in E. coli to investigate the ribosome-associated chaperone network and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL and ClpB contribute increasingly, when DnaK is deficient. Surprisingly, misfolding of NCs due to defects in co-translational chaperone function or amino acid analog incorporation, results in recruitment of the non-canonical release factor RF3 to the ribosome. RF3 then cooperates with RF2 in mediating premature chain termination of a subset of abundant NCs, allowing their efficient degradation. This function of RF3 reduces the accumulation of misfolded proteins. It is critical for proteostasis maintenance and cell growth under conditions of limited chaperone availability.
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Project description:As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, β-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors. Examination of translation in the Gram-negative bacterium Escherichia coli, as well as an analysis of the interactions between nascent chains and the molecular chaperone Trigger Factor.
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Project description:The acquisition of multi-drug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the non-pathogenic α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (β-lactam) antibiotics. Combining two-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR targets a common operator of divergent tipR and acrAB-nodT promoter in adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified compounds that either interfere with DNA-binding by TipR or induce its ClpXP-dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT disfigures the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, to boost pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.
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Project description:The acquisition of multi-drug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the non-pathogenic α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (β-lactam) antibiotics. Combining two-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR targets a common operator of divergent tipR and acrAB-nodT promoter in adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified compounds that either interfere with DNA-binding by TipR or induce its ClpXP-dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT disfigures the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, to boost pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.
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Project description:As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, β-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors.
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