Ribosome-bound Nascent Chains Research Articles

Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1–140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63–94 residues. Longer RNCs (apoHmp1–189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.

Mapping Protein-Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome-Nascent Globin Complex.

Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein-ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.

Resolving chaperone action on ribosome-bound nascent chains with single-molecule spectroscopy

Resolving chaperone action on ribosome-bound nascent chains with single-molecule spectroscopy

Nascent-chain interaction networks and their effect on the bacterial ribosome.

In order to become bioactive, proteins need to be biosynthesized and protected from aggregation during translation. Remarkably, the ribosome contributes to both of these tasks. Some ribosomal proteins (r-proteins) are known to noncovalently interact with ribosome-bound nascent chains (RNCs). Yet, specific interaction networks and their role within the ribosomal machinery remain poorly characterized. Here, we found that empty ribosomes are more sensitive to urea denaturation than ribosomes harboring nascent protein chains, including aminoacyl or short peptidyl-tRNA (snc-tRNA). This result suggests that snc-tRNAs play a ribosome-stabilizing role. On the other hand, RNCs of variable sequence and length do not significantly modify the apparent stability of the peptidyl-transferase center (PTC) and r-proteins, suggesting that RNC/r-protein interaction networks close to the PTC have a negligible effect on the apparent ribosome stability. Fluorescence anisotropy decay, chemical-crosslinking and Western blots show that 140-residue-long RNCs of the foldable protein apoHmp have an N-terminal compact region (63-94 residues) and interact with r-protein L23 but not with L24 or L29. Longer RNCs bear a similar compact region and interact either with L23 or with L23 and another unidentified r-protein or with molecular chaperones. Interestingly, the apparent stability of RNC/r-protein complexes does not depend on RNC sequence or length. Taken together, our findings show that RNCs establish preferential interactions with the L23 ribosomal protein whose strength does not depend on RNC amino-acid sequence and length. As the nascent chain elongates, the interaction network extends to additional r-proteins and chaperones. It follows that the ribosome provides uniform, indiscriminate support to nascent proteins regardless of their sequence and foldability. This result highlights the mode of action of this unbiased molecular machine for cellular health.

Kinetic control of nascent protein biogenesis by peptide deformylase

Synthesis of bacterial proteins on the ribosome starts with a formylated methionine. Removal of the N-terminal formyl group is essential and is carried out by peptide deformylase (PDF). Deformylation occurs co-translationally, shortly after the nascent-chain emerges from the ribosomal exit tunnel, and is necessary to allow for further N-terminal processing. Here we describe the kinetic mechanism of deformylation by PDF of ribosome-bound nascent-chains and show that PDF binding to and dissociation from ribosomes is rapid, allowing for efficient scanning of formylated substrates in the cell. The rate-limiting step in the PDF mechanism is a conformational rearrangement of the nascent-chain that takes place after cleavage of the formyl group. Under conditions of ongoing translation, the nascent-chain is deformylated rapidly as soon as it becomes accessible to PDF. Following deformylation, the enzyme is slow in releasing the deformylated nascent-chain, thereby delaying further processing and potentially acting as an early chaperone that protects short nascent chains before they reach a length sufficient to recruit other protein biogenesis factors.

An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel

The influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.

High-Resolution Insights into Nascent-Chain Conformation by Single-Particle Cryo-Electron Microscopy

The folding of nascent proteins emerging out of the ribosomal exit tunnel is poorly understood. Progress is needed in this area because the conformation of ribosome-bound nascent chains (RNCs) likely affects the balance between protein folding and deleterious aggregation early in protein life. In this work, we employ the phosphorylated-insulin-receptor-interacting region of the growth factor protein 14 (Grb14) from Rattus norvegicus, also known as PIR, to explore its structural features by single-particle cryo-electron microscopy (sp-cryo-EM).

Timing and Specificity of Cotranslational Nascent Protein Modification in Bacteria

Selection of newly‐synthesized proteins into correct protein biogenesis pathways is crucial for cellular homeostasis. The ubiquitous N‐terminal methionine excision (NME) process is mediated by peptide deformylase (PDF) and methionine aminopeptidase (MAP), two essential enzymes in bacteria. This reaction takes place near the nascent peptide exit site of the ribosome, where multiple ribosome‐associating protein biogenesis factors (RPBs) also compete for the access to the nascent chain. How NME achieves its efficiency and specificity at this crowded environment is unknown. Here, using kinetic measurements on purified ribosome‐nascent chain complexes, we show that the ribosome accelerates the MAP reaction for optimal substrates by 102–104 folds. Kinetic competition with translation elongation and selective regulation from other RPBs enhance the specificity of NME by narrowing the processing time window for reactions on suboptimal substrates. With the kinetic data, we constructed a mathematical model and accurately predicted the cotranslational NME efficiency in cytosol. Our data demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity. Moreover, the remodeled MAP activity prompted us to develop a cotranslational assay to screen for MAP inhibitors in the physiological context of translating ribosome. The results explain the discrepancy between traditional peptide‐based assays and cellular data, providing a powerful tool for the development of antibacterial agents targeting the NME machineries.Support or Funding InformationThis work was supported by National Institutes of Health grant GM078024 and a grant from the Weston Havens Foundation to S.‐o. Shan, and a Think Global Education Trust Fellowship from Taiwan to C.‐I. Yang.

Ribopuromycylation in Coronavirus-Infected Cells.

Ribopuromycylation enables the visualization and quantitation of translation on a cellular level by immunofluorescence or in total using standard western blotting. This technique uses ribosome catalyzed puromycylation of nascent chains followed by immobilization on the ribosome by antibiotic chain elongation inhibitor emetine. Detection of puromycylated ribosome-bound nascent chains can then be achieved using a puromycin-specific antibody.

Timing and specificity of cotranslational nascent protein modification in bacteria

The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.

Non-equilibrium dynamics of a nascent polypeptide during translation suppress its misfolding

Protein folding can begin co-translationally. Due to the difference in timescale between folding and synthesis, co-translational folding is thought to occur at equilibrium for fast-folding domains. In this scenario, the folding kinetics of stalled ribosome-bound nascent chains should match the folding of nascent chains in real time. To test if this assumption is true, we compare the folding of a ribosome-bound, multi-domain calcium-binding protein stalled at different points in translation with the nascent chain as is it being synthesized in real-time, via optical tweezers. On stalled ribosomes, a misfolded state forms rapidly (1.5 s). However, during translation, this state is only attained after a long delay (63 s), indicating that, unexpectedly, the growing polypeptide is not equilibrated with its ensemble of accessible conformations. Slow equilibration on the ribosome can delay premature folding until adequate sequence is available and/or allow time for chaperone binding, thus promoting productive folding.

Nascent Proteins Interact with Key Regions of the Outer Surface of the Ribosome

While in vitro protein folding is reasonably well understood, very little is known about how nascent chains achieve their native state in the complex cellular environment. The earliest stages of protein folding as nascent chains emerge out of the ribosome are still poorly characterized. Proteins and rRNA located within the ribosomal exit tunnel and outer surface, for instance, may interact with nascent proteins and affect their folding. Previous studies in the Cavagnero group showed that ribosome-bound nascent chains (RNCs) derived from the intrinsically disordered protein PIR and its highly negatively charged variants exhibit a significant spatial bias, consistent with the presence of non-covalent interactions between RNCs and the ribosomal surface. In this work, we probe RNC-ribosomal-protein contacts by a combination of chemical crosslinking and Western blotting, and show direct evidence for well-defined interactions between RNCs of PIR and specific ribosomal proteins. Surprisingly, the interacting ribosomal proteins are clustered only on one side of the ribosomal surface, relative to the exit tunnel. The observed interactions are particularly strong when about 15 amino acids have emerged outside of the ribosomal exit tunnel, and get weaker as the chain gets longer. This result suggests that the interactions are targeted towards the prevention of co-translational aggregation. This work corroborates an emerging view according to which the ribosome plays a fundamental role in protein biogenesis that extends well beyond the mere catalysis of peptide bond formation.

Yeast molecular chaperone gene SSB2 is involved in the endoplasmic reticulum stress response.

The Saccharomyces cerevisiae chaperone gene SSB2 belongs to the Hsp70 family. Unlike other HSP70 genes, SSB2 gene expression is reduced after heat shock. It has been reported that Ssb2p can be cross-linked to ribosome-bound nascent polypeptide chains, suggesting a potential role of SSB2 in the endoplasmic reticulum (ER) stress response. In this study, SSB2-deletion and SSB2-overexpression yeast strains were generated and applied to explore the potential mechanism by which SSB2 is involved in the tunicamycin (TM)-induced ER stress response. We demonstrate for the first time that SSB2 deficiency results in reduced resistance to TM, while overexpression of SSB2 increases resistance to TM in an IRE1-HAC1 pathway-dependent manner; theseobservationsarerelatedto changes in intracellular unfolded protein response activities (under the TM-stressed condition). Additionally, SSB2 deletion induces early apoptosis and it may play a causal role in the shortened replicative life span of ssb2Δ mutants observed in this study. These findings highlight the involvement of SSB2 in ER stress responses and ageing in yeast.

Novel in‐vitro tag‐and‐modify protein sample generation methods for multiplexed singlemolecule FRET screening

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we extend our recent work demonstrating that combinations of purified and reconstituted in-vitro translation, quantitative unnatural amino acid incorporation, and copper-catalyzed azide-alkyne cycloaddition can overcome these challenges. In particular, we demonstrate the ability to quantitatively label azide or alkyne-tagged proteins and ribosome-bound nascent polypeptide chains (RNCs) with pairs of dyes with greater flexibility and more specificity than was previously possible. This sample generation approach can provide protein samples suitable for highly-multiplexed single-molecule FRET-based conformational phenotyping. Importantly, dual-labeled RNC libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and can enable the in-vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest. Support or Funding Information California State University Program for Education and Research in Biotechnology New Investigator Award (Kambiz M. Hamadani) Ribosome-bound nascent polypeptide chains labeled with pairs of dyes for multiplexed single molecule FRET-based screening This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expanding the Scope of Single Molecule FRET Spectroscopy towards Primarily Undergraduate Institutions

Single molecule fluorescence methods provide a unique and ultrasensitive set of probes for monitoring the conformations and dynamics of protein chains within physiologically relevant contexts. In particular, single molecule fluorescence resonance energy transfer (smFRET) is capable of monitoring the distances between two fluorescent dyes with sub-nanometer spatial and nanosecond temporal resolution. Unfortunately, smFRET remains inaccessible to many biophysical researchers and most of all to students at primarily undergraduate research institutions. This is in part due to the difficulties involved in making dual-labelled protein samples but also stems from the expense and/or complexity of existing single-molecule fluorescence detection platforms. To address the first issue, we have developed a sample generation protocol suitable for making large libraries of dual-labelled proteins and ribosome-bound nascent chains (RNCs). This method consists of using a purified and reconstituted in-vitro translation system for protein expression, the incorporation of azide or alkyne-bearing non-standard amino acids, and finally click-chemistry-based dye attachment. Here, we show that this approach is simple and robust enough to be used by undergraduate students to make large libraries of dual-labelled protein samples in about a day. To enable smFRET-based screening of these libraries we have also built the first-ever confocal smFRET microscope at a primarily undergraduate institution. This instrument was built for less than $20K largely using second-hand optical components. It has a 3-axis piezo-scanning stage which enables single-molecule imaging and trajectory analysis on surface-immobilized molecules. It also has alternating laser excitation capabilities which enable digital sorting of single molecules using multi-dimensional histograms. This work marks the first implementation of smFRET as a research tool at a primarily undergraduate research institution.

Rapidly Translated Polypeptides Are Preferred Substrates for Cotranslational Protein Degradation

Nascent polypeptides are degraded by the proteasome concurrently with their synthesis on the ribosome. This process, called cotranslational protein degradation (CTPD), has been observed for years, but the underlying mechanisms remain poorly understood. Equally unclear are the identities of cellular proteins genuinely subjected to CTPD. Here we report the identification of CTPD substrates in the yeast Saccharomyces cerevisiae via a quantitative proteomic analysis. We compared the abundance of individual ribosome-bound nascent chains between a wild type strain and a mutant defective in CTPD. Of 1,422 proteins acquired from the proteomic analysis, 289 species are efficient CTPD substrates, with >30% of their nascent chains degraded cotranslationally. We found that proteins involved in translation, ribosome biogenesis, nuclear transport, and amino acid metabolism are more likely to be targeted for CTPD. There is a strong correlation between CTPD and the translation efficiency. CTPD occurs preferentially to rapidly translated polypeptides. CTPD is also influenced by the protein sequence length; longer polypeptides are more susceptible to CTPD. In addition, proteins with N-terminal disorder have a higher probability of being degraded cotranslationally. Interestingly, the CTPD efficiency is not related to the half-lives of mature proteins. These results for the first time indicate an inverse correlation between CTPD and cotranslational folding on a proteome scale. The implications of this study with respect to the physiological significance of CTPD are discussed.

Nascent Proteins Interact with Key Regions of the Outer Surface of the Ribosome

The initial stages of a protein's life in the cell are crucial for the proper balance between folding, misfolding and aggregation. While an appreciable amount of information is known about protein folding/unfolding after biosynthesis, we lack a clear understanding of how ribosome bound nascent chains (RNCs) acquire any degree of structure and dynamics. Previous studies in the Cavagnero group showed that RNCs of the intrinsically disordered protein PIR (i.e., the phosphorylated insulin receptor) and its highly negatively charged mutant PIR 1.2.3 exhibit significant spatial bias towards the ribosomal surface. We now present findings that pinpoint the presence of well-defined interactions between RNCs of PIR, PIR 1.2.3, as well as the foldable proteins bacterial flavohemoglobin apoHmpH and sperm whale apomyoglobin, with specific ribosomal proteins and the co-translationally active chaperone trigger factor (TF). RNC-ribosome contacts were probed by chemical crosslinking followed by mass spectrometry analysis, and nascent protein local and global dynamics were assessed by time-resolved fluorescence depolarization. Initial results indicate that under physiologically relevant conditions RNCs of both PIR and apoHmpH crosslink to TF in a strategic manner. In addition to interactions with TF, multiple populations of apoHmpH RNCs were observed to transiently bind to specific proteins located on the outer surface of the ribosome, both near and far from the exit tunnel. We are currently exploring the implications of these specific interactions on the balance between protein folding and aggregation. Interestingly, these interactions are strongly Mg2+ concentration-dependent. The solubility profile of apoHmpH upon release from the ribosome is not Mg2+ concentration-dependent, suggesting that the above interactions may play a yet-unidentified regulatory role in translation.

Nonorthogonal tRNA(cys)(Amber) for protein and nascent chain labeling.

In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNAcysAmber from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNAcysAmber with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNAcysAmber, which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.

A comparison of the folding characteristics of free and ribosome‐tethered polypeptide chains using limited proteolysis and mass spectrometry

The kinetics and thermodynamics of protein folding are commonly studied in vitro by denaturing/renaturing intact protein sequences. How these folding mechanisms relate to de novo folding that occurs as the nascent polypeptide emerges from the ribosome is much less well understood. Here, we have employed limited proteolysis followed by mass spectrometry analyses to compare directly free and ribosome-tethered polypeptide chains of the Src-homology 3 (SH3) domain and its unfolded variant, SH3-m10. The disordered variant was found to undergo faster proteolysis than SH3. Furthermore, the trypsin cleavage patterns observed show minor, but significant, differences for the free and ribosome-bound nascent chains, with significantly fewer tryptic peptides detected in the presence of ribosome. The results highlight the utility of limited proteolysis coupled with mass spectrometry for the structural analysis of these complex systems, and pave the way for detailed future analyses by combining this technique with chemical labeling methods (for example, hydrogen-deuterium exchange, photochemical oxidation) to analyze protein folding in real time, including in the presence of additional ribosome-associated factors.

Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.