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1. Nascent-Chain Interaction Networks and Their Effect on the Bacterial Ribosome

   Masse, Meranda M; Guzman-Luna, Valeria; Varela, Angela E; Hutchinson, Rachel B; Srivastava, Aniruddha; Wei, Wanting; Fuchs, Andrew M; Cavagnero, Silvia (November 2022, Bioarchives (BioRXiv))

   N.A. (Ed.)

   In order to become bioactive, proteins need to be biosynthesized and protected from aggregation during translation. The ribosome and molecular chaperones contribute to both tasks. While it is known that some ribosomal proteins (r-proteins) interact with ribosome-bound nascent chains (RNCs), specific interaction networks and their role within the ribosomal machinery remain poorly characterized and understood. Here, we find that RNCs of variable sequence and length (beyond the 1st C-terminal reside) do not modify the apparent stability of the peptidyl-transferase center (PTC) and r-proteins. Thus, RNC/r-protein interaction networks close to the PTC have no effect on the apparent stability of ribosome-RNC complexes. Further, fluorescence anisotropy decay, chemical-crosslinking and Western blots show that RNCs of the foldable protein apoHmp1-140 have an N-terminal compact region (6394 residues) and interact specifically with r-protein L23 but not with L24 or L29, at the ribosomal-tunnel exit. Longer RNCs bear a similar compact region and interact either with L23 alone or with L23 and another unidentified r-protein, or with molecular chaperones. The apparent strength of RNC/r-protein interactions does not depend on RNC sequence. Taken together, our findings show that RNCs encoding foldable protein sequences establish an expanding specific interaction network as they get longer, including L23, another r-protein and chaperones. Interestingly, the ribosome alone (i.e., in the absence of chaperones) provides indiscriminate support to RNCs bearing up to ca. 190 residues, regardless of nascent-chain sequence and foldability. In all, this study highlights the unbiased features of the ribosome as a powerful nascent-protein interactor. 

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

   https://doi.org/10.1038/s42003-021-02752-4  

   Guzman-Luna, Valeria; Fuchs, Andrew M.; Allen, Anna J.; Staikos, Alexios; Cavagnero, Silvia (October 2021, Communications Biology)

   Abstract 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. 

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3. Mg+2 Ions Mediate the Interaction of Intrinsically Disordered Nascent Chains with the Ribosome

   Cavagnero, Silvia and (March 2022, The neuroscience chronicles)

   During the last few decades, the ribosome has been regarded primarily as a major cell player devoted to the catalysis of protein biosynthesis during translation [1-5]. It is therefore not surprising that several processes related to translation exploit the ribosome as a central hub. For instance, it is well-known that many events related to translational regulation are mediated by interactions between the ribosome and initiation, elongation or termination factors [6-9]. In addition, the ribosome is involved in mRNA-code recognition and proofreading [10-12] as well as in the control of translation rates via interactions with mRNA codons bearing high- and lowfrequency [13-15] and associated with variable tRNA abundance within the translation machinery [16-18]. Interestingly, the ribosome also assists de novo protein structure formation by minimizing cotranslational aggregation, thus increasing the yield of native-protein production [19,20]. The latter event, however, has not been shown to require -- or even involve -- direct interactions between the ribosome and the nascent protein chain. A notable exception is that of nascent chains bearing either N-terminal signal sequences or translational-arrest tags. These proteins are known to establish short- or long-term contacts with various regions of the ribosome during translation [21-25]. In summary, until recently very little knowledge has been available about direct contacts between the ribosome and nascent polypeptides and proteins that do not carry signal or arrest sequences. Studies based on fluorescence depolarization in the frequency domain [26] and NMR spectroscopy [27-30] provided interesting data that are consistent with, but do not unequivocally establish, the presence of these interactions. 

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4. Proteome-wide determinants of co-translational chaperone binding in bacteria

   https://doi.org/10.1038/s41467-025-59067-9  

   Galmozzi, Carla Verónica; Tippmann, Frank; Wruck, Florian; Auburger, Josef Johannes; Kats, Ilia; Guennigmann, Manuel; Till, Katharina; O´Brien, Edward P; Tans, Sander J; Kramer, Günter; et al (December 2025, Nature Communications)

   Abstract 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 mainE. colichaperones 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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5. Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation

   https://doi.org/10.1021/acssynbio.3c00357  

   Steinkühler, Jan; Peruzzi, Justin A; Krüger, Antje; Villaseñor, Citlayi G; Jacobs, Miranda L; Jewett, Michael C; Kamat, Neha P (January 2024, ACS Synthetic Biology)

   Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE. 

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