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

Abstract

In order to become bioactive, proteins need to be biosynthesized and protected from aggregation during translation. The ribosome and molecular chaperones contribute to both of these 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.

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

“…The presence of noncovalent contacts between RNCs and the L23 r-protein were previously detected for the nascent intrinsically disordered protein (IDP) PIR and for the nascent E. coli globin apoHmpH via a combination of EDC cross-linking and Western blotting. Here, we find that apoMb RNCs display an EDC-cross-linking gel-band pattern qualitatively similar to those of apoHmpH and PIR, as shown in Figure d.…”

“…While chemical cross-linkers have been previously used to facilitate 3D structure determination of dynamic macromolecules, this approach has not been utilized for RNCs. The zero-length chemical cross-linker EDC has previously been used to identify the interaction of r-proteins with nascent chains, , rendering it an ideal candidate for cross-linking dynamic RNCs to the ribosomal surface. Given that (i) nascent chains only cross-link when they are in close proximity to the ribosome, (ii) prior kinetic studies showed that the majority of nascent chain and r-protein interactions occur very quickly, and (iii) EDC tends to provide an underestimate of the actual populations of interacting RNCs, it is appropriate to regard the cross-linked structure presented here as appropriately capturing the presence of RNC–r-protein contacts.…”

“…Previous studies showed that the interaction of full-length nascent E. coli globin domains with ribosomal proteins are mostly displaced by interactions with TF at physiologically relevant TF concentrations (ca. 8–20 μM) in purified RNCs resuspended in buffer . On the other hand, competition for TF is expected to be fierce within the complex live-cell environment of E. coli , given the many types of RNCs present in the cytosol, the comparable TF and active ribosome concentrations, the fact that multiple TF molecules may bind single RNCs, and the variable affinity of TF for nascent protein chains. , Therefore, we expect that some RNCs are TF-free in the cellular environment.…”

“…Conversely, in the presence of nascent chains bearing N-terminal membrane-tagging signal sequences, proximity with L23, L24, and L29 r-proteins has been detected in the tunnel vestibule, ,,, though the authors did not explicitly quote the presence of actual interactions with these r-proteins. In the case of nascent chains lacking N- or C-terminal signal or stalling sequences, chemical cross-linking revealed the presence of interactions with specific ribosomal proteins (r-proteins) L23 and L29 (for ribosome-bound nascent chains (RNCs) of an intrinsically disordered protein) or only with L23 (for RNCs of E. coli apoHmpH, a foldable single-domain globin) …”

“…In the case of nascent chains lacking N-or C-terminal signal or stalling sequences, chemical cross-linking revealed the presence of interactions with specific ribosomal proteins (r-proteins) L23 and L29 (for ribosome-bound nascent chains (RNCs) of an intrinsically disordered protein) 45 or only with L23 (for RNCs of E. coli apoHmpH, a foldable single-domain globin). 31 Despite all of the above knowledge, which highlights the vital role played by the ribosome in protein biosynthesis and folding, crucial information on the specific r-protein and rRNA interaction sites, especially across the vestibule and outer ribosomal surface, is missing. Further, the nature of the ribosome−RNC interactions is not clear.…”