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SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57-98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57-98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest., (© 2020 The Author(s).)

SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain outside the ribosome affects the stability of translation arrest. To clarify this issue, we performed in vitro translation assays with HaloTag proteins fused to the C-terminal fragment of E. coli SecM containing the arrest sequence or the full-length SecM. We showed that the translation of HaloTag proteins, which are fused to the fragment, is not effectively arrested, whereas the translation of HaloTag protein fused to full-length SecM is arrested efficiently. In addition, we observed that the nascent SecM chain outside the ribosome markedly stabilizes the translation arrest. These results indicate that changes in the nascent polypeptide chain outside the ribosome can affect the stability of translation arrest; the nascent SecM chain outside the ribosome stabilizes the translation arrest.

The dynamics of full protein synthesis and the co-translational folding processes are not fully understood. We have developed a novel method, using a combination of ribosome display and single-molecule techniques, for monitoring the synthesis, co-translational folding, and maturation of a complete polypeptide chain at the single-molecule level. This method enabled us to observe the appearance of green fluorescent protein fluorescence after de novo synthesis of the complete protein. Here, we provide the information necessary to reproduce this method, which will be valuable in revealing the dynamics of the co-translational folding and maturation of nascent polypeptides.

How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro. Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombinant Elements that lacked ribosomes but contained purified factors and enzyme that are required for translation in Escherichia coli. Translation was monitored using a GFP mutant (F64L/S65T/F99S/M153T/V163A) that has a high fluorophore maturation rate and that contained the Secretion Monitor arrest sequence to prevent dissociation from the ribosome. Immobilized ribosomal subunits were labeled with Cy3 and GFP synthesis was measured by colocalization of GFP fluorescence with the ribosome position. The rate of appearance of colocalized ribosome GFP was equivalent to the rates of fluorescence appearance coupled with translation measured in bulk, and the ribosome-polypeptide complexes were stable for hours. The methods presented here are applicable to single-molecule investigation of translational initiation, elongation and cotranslational folding.