Biology subjects

Chadani, Y.

Publications and source records attributed to Chadani, Y..

8 recordsLinked to original sources

bioRxiv2025

Structural insights into YheS-mediated release of SecM-arrested ribosome.

ATPase-binding cassette subfamily F (ABCF) proteins interact with the ribosome to resolve translation defects near the peptidyl-transferase center, induced by ribosome-targeting antibiotics or "hard-to-translate" nascent peptides. In Escherichia coli, four ABCF proteins resolve the translation of distinct problematic sequences, but their molecular mechanisms remain unclear. Here, we present a 2.8-[A] cryo-EM structure of the SecM-stalled ribosome in complex with an ATPase-deficient mutant of YheS, an ABCF protein that releases ribosomes stalled during the translation of SecM, a representative ribosome-arresting peptide. YheS is bound to the E-site and relocates the P-site tRNA (P-tRNA) by displacing its CCA-end. Notably, the density of the SecM nascent chain mostly disappeared upon YheS binding. Molecular dynamics simulations support that the P-tRNA relocation generates a pulling force that disrupts key intratunnel interactions essential for arrest. This mechanism is functionally analogous to, yet mechanistically distinct from, the translocon-mediated external pulling force for SecM release. TeaserThe release mechanism of the SecM-arrested ribosome by the ABCF protein YheS was elucidated by structure-based analyses.

biochemistry↗

bioRxiv2024

A newly-identified mini-hairpin shaped nascent peptide blocks translation termination by a novel mechanism.

Protein synthesis by ribosomes not only produces functional proteins but also serves diverse functions depending on the coding amino acid sequences. Certain nascent peptides interact with the ribosome exit tunnel to arrest translation and modulate the expression of downstream genes or themselves. However, a comprehensive understanding of the mechanisms of such ribosome stalling and its regulation remains elusive. In this study, we systematically screened for unidentified ribosome arrest peptides through phenotypic evaluation, proteomics, and MS analyses, leading to the discovery of novel arrest peptides PepNL and NanCL in E. coli. Our cryo-EM study on PepNL revealed a unique arrest mechanism, in which the N-terminus of PepNL folds back towards the tunnel entrance to prevent the catalytic GGQ motif of release factor from accessing the peptidyl transferase center, causing translation arrest at the UGA stop codon. Furthermore, unlike other sensory arrest peptides that require an arrest inducer, PepNL uses tryptophan as an arrest releaser, where Trp-tRNA reads through the stop codon. Our findings illuminate the mechanism and regulatory framework of nascent peptide-induced translation arrest, paving the way for exploring regulatory nascent peptides.

molecular biology↗

bioRxiv2023

The ABCF proteins in Escherichia coli individually alleviate nascent peptide-induced noncanonical translations.

Organisms possess a wide variety of proteins with a diverse repertoire of amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the translation elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors, enabling the synthesis of diverse proteins. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various noncanonical translations induced by nascent peptide sequences within the exit tunnel of the ribosome. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent ribosome stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that the ATP hydrolysis-coupled structural rearrangement and interdomain linker sequence between the two nucleotide-binding domains play crucial roles in alleviating the noncanonical translations. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences. Significance statementProteins, that constitute living organisms, exhibit a diverse range of amino acid sequences. However, it has become evident that ribosomes have difficulties in synthesizing certain amino acid sequences, including the poly-basic, poly-acidic, and poly-proline sequences. The mechanisms underlying the expression of proteins with such challenging sequences remain largely elusive. In this study, we have unveiled that translation factor ABCF proteins in Escherichia coli promote various kinds of problematic amino acid sequences that inhibit efficient translation. Through the actions of translation elongation factors including the ABCF proteins, the translation system acquires robustness in synthesizing a vast repertoire of amino acid sequences.

biochemistry↗

bioRxiv2022

Novel trans-translation-associated gene regulation revealed by prophage excision-triggered switching of ribosome rescue pathway

Escherichia coli has multiple pathways to release a nonproductive ribosome complex stalled at the 3 end of nonstop mRNAs: SsrA RNA-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and thus its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the situations in which ArfA is expressed to rescue the nonproductive complexes have not been elucidated. Here, we demonstrated that the excision of the CP4-57 prophage adjacent to the E. coli ssrA gene leads to the inactivation of SsrA RNA and switches the primary rescue pathway from trans-translation to the ArfA/RF2 pathway. A comparative quantitative proteomic analysis revealed that the switching of the rescue pathway rearranges not only the proteome landscape in E. coli cells but also the phenotype, such as motility. Among the proteins with significantly increased abundance in the ssrA-inactivated cells, we found ZntR, whose mRNA is transcribed together as the upstream part of the nonstop arfA mRNA. Further analysis revealed that the translation of the nonstop ORF of arfA triggered the repression of the upstream zntR ORF, via trans-translation-coupled exonucleolytic (RUTEX) degradation by a polynucleotide phosphorylase. These results provide a novel example of trans-translation-dependent regulation, and shed new light on the physiological roles of prophages in gene expression.

genetics↗

bioRxiv2022

A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains; i.e., polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified [~]800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods for profiling nascent chains such as ribosome profiling. Significance StatementIn the central dogma of biology, RNA and protein are usually regarded as two completely independent molecular species. However, they are combined into a single species called peptidyl-tRNA (pep-tRNA) during the translation process in the ribosome. Despite the importance of pep-tRNAs as precursors of all proteins in the cell, a general method to analyze pep-tRNAs on a large scale was lacking. Taking advantage of the properties of pep-tRNAs as RNA and protein, we developed a method to enrich the pep-tRNAs by organic solvent extraction and silica column separation. The method, termed PETEOS, not only provides a unique approach to examine the nascent state of proteins but also may be effective in capturing snapshots of translation status in the cell.

biochemistry↗

bioRxiv2022

Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes

Robust translation elongation of any given amino acid sequence is a prerequisite to shape proteomes. Nevertheless, nascent peptides could destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains stochastically can induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in the N-terminal regions could prematurely terminate translation, producing premature products as peptidyl-tRNA species. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we found many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk. Indeed, a bioinformatics analysis revealed that the N-terminal regions of ORFs avoid D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

biochemistry↗

bioRxiv2021

Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. Negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that the risk of IRD is maximal at the N-terminal regions of proteins encoded by dozens of Escherichia coli genes. In contrast, most potential IRD sequences in the middle of open reading frames remain cryptic. We found two elements in nascent chains that counteract IRD: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

biochemistry↗

bioRxiv2020

Escherichia coli small heat shock protein IbpA is an aggregation-sensor that self-regulates its own expression at a translational level

Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps in Escherichia coli, is regulated by the {sigma}32 heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or the ibpA gene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsp expression. Oligomeric IbpA self-represses the ibpA/ibpB expression at the translational level, but the self-repression is relieved by the sequestration of IbpA into protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation. Author summaryAll organisms have protein quality control systems against stresses disturbing cellular protein homeostasis (proteostasis). The systems have multiple stages: folding, degradation, and sequestration. Sequestration of denatured proteins is the first step to support other maintenance strategies. Small heat shock proteins (sHsps), which are well-conserved chaperones, are representative "sequestrases" that co-aggregate with denatured proteins. We found that IbpA, an Escherichia coli sHsp, is a direct mediator for negative feedback regulation at the translational level. Recruitment of IbpA into the protein aggregates relieves the ibpA expression suppression. This novel mechanism of IbpA as an aggregation-sensor tightly regulates the IbpA level, enabling the sHsp to function as a sequestrase upon aggregation stress.

biochemistry↗