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

   https://doi.org/10.1038/s41598-024-61274-1  

   Masse, Meranda_M; Guzman-Luna, Valeria; Varela, Angela_E; Mahfuza_Shapla, Ummay; Hutchinson, Rachel_B; Srivastava, Aniruddha; Wei, Wanting; Fuchs, Andrew_M; Cavagnero, Silvia (May 2024, Scientific Reports)

   Abstract 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 apoHmp_1–140interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63–94 residues. Longer RNCs (apoHmp_1–189) 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. 

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3. The ribosome: A hot spot for the identification of new types of protein methyltransferases

   https://doi.org/10.1074/jbc.AW118.003235  

   Clarke, Steven G. (January 2018, Journal of Biological Chemistry)

   Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for posttranslationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, while the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins. 

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4. Minimization of the E. coli ribosome, aided and optimized by community science

   https://doi.org/10.1093/nar/gkad1254  

   Tangpradabkul, Tiyaporn; Palo, Michael; Townley, Jill; Hsu, Kenneth B; participants, Eterna; Smaga, Sarah; Das, Rhiju; Schepartz, Alanna (January 2024, Nucleic Acids Research)

   Abstract The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome. 

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5. 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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