VemP is a secretory protein in the Vibrio species that monitors cellular protein-transport activity through its translation arrest, allowing expression of the downstream secD2-secF2 genes in the same operon, which encode components of the protein translocation machinery. When cellular protein-transport function is fully active, secD2/F2 expression remains repressed as VemP translation arrest is canceled immediately. The VemP arrest-cancellation occurs on the SecY/E/G translocon in a late stage in the translocation process and requires both trans-factors, SecD/F and PpiD/YfgM, and a cis-element, Arg-85 in VemP; however, the detailed molecular mechanism remains elusive. This study aimed to elucidate how VemP passing through SecY specifically monitors SecD/F function. Genetic and biochemical studies showed that SecY is involved in the VemP arrest-cancellation and that the arrested VemP is stably associated with a specific site in the protein-conducting pore of SecY. VemP-Bla reporter analyses revealed that a short hydrophobic segment adjacent to Arg-85 plays a critical role in the regulated arrest-cancellation with its hydrophobicity correlating with the stability of the VemP arrest. We identified Gln-65 and Pro-67 in VemP as novel elements important for the regulation. We propose a model for the regulation of the VemP arrest cancellation by multiple cis-elements and trans-factors with different roles.

Ribosomes polymerize nascent peptides through repeated inter-subunit rearrangements between the classic and hybrid states. The peptidyl-tRNA, the intermediate species during translation elongation, stabilizes the translating ribosome to ensure robust continuity of elongation. However, the translation of acidic residue-rich sequences destabilizes the ribosome, leading to a stochastic premature translation cessation termed intrinsic ribosome destabilization (IRD), which is still ill-defined. Here, we dissect the molecular mechanisms underlying IRD in Escherichia coli. Reconstitution of the IRD event reveals that (1) the prolonged ribosome stalling enhances IRD-mediated translation discontinuation, (2) IRD depends on temperature, (3) the destabilized 70S ribosome complex is not necessarily split, and (4) the destabilized ribosome is subjected to peptidyl-tRNA hydrolase-mediated hydrolysis of the peptidyl-tRNA without subunit splitting or recycling factors-mediated subunit splitting. Collectively, our data indicate that the translation of acidic-rich sequences alters the conformation of the 70S ribosome to an aberrant state that allows the noncanonical premature termination.

The formation of protein complexes is crucial to most biological functions. The cellular mechanisms governing protein complex biogenesis are not yet well understood, but some principles of cotranslational and posttranslational assembly are beginning to emerge. In bacteria, this process is favored by operons encoding subunits of protein complexes. Eukaryotic cells do not have polycistronic mRNAs, raising the question of how they orchestrate the encounter of unassembled subunits. Here we review the constraints and mechanisms governing eukaryotic co- and posttranslational protein folding and assembly, including the influence of elongation rate on nascent chain targeting, folding, and chaperone interactions. Recent evidence shows that mRNAs encoding subunits of oligomeric assemblies can undergo localized translation and form cytoplasmic condensates that might facilitate the assembly of protein complexes. Understanding the interplay between localized mRNA translation and cotranslational proteostasis will be critical to defining protein complex assembly in vivo.

Proteins are biological macromolecules essential for cells to maintain their metabolic activities. Proteins are synthesized during translation elongation, a synergistic process in which ribosomes decode the genetic information transmitted in mRNA, using tRNA. Numerous human diseases, such as neurodegenerative diseases and cancers, are known to be related to abnormal translation elongation. Translation elongation, as one of the two critical steps for the central dogma, used to be the focus of research in molecular biology. However, limitations in methodology had hindered further investigations on the dynamic process of translation elongation and its regulation. Recently, breakthroughs in methodology have revived this research field. Studies in the past decade or so have revealed that, beyond simple decoding of genetic information in mRNA, translation elongation entails sophisticated regulatory mechanisms and multifaceted biological consequences; such insights have provided a novel theoretical framework for understanding the maintenance of protein homeostasis and the development of diseases. In this review, we summarize the most updated methods that can be used to investigate the processes of translation elongation and highlight the mechanisms by which mRNA and protein sequences modulate the local rate of translation elongation. We further enumerate the consequences of dysregulation in translation elongation, from various aspects such as mRNA stability, protein synthesis and degradation, protein subcellular localization, and co-translational protein folding. We anticipate that this review will serve to draw the attention of scholars in various research fields to participate in the study of translation elongation.
翻译延伸是核糖体将信使RNA (mRNA)蕴含的遗传信息解码为蛋白质的有序过程,是细胞维持基本代谢活动的核心步骤。多种人类疾病(如神经退行性疾病、癌症等)都与翻译延伸的异常有关。翻译延伸作为中心法则的关键步骤曾是现代分子生物学研究的重点内容,然而方法学上的限制却阻碍了对其动态过程以及调控规律的进一步研究。近年来,对翻译延伸调控相关方法的突破让与其相关的生命科学研究获得了长足的发展,尤其是近10年来的研究揭示了翻译延伸的复杂调控机理和多种生物学效应,为理解蛋白表达调控和疾病发生的关联提供了新的理论视角。本文在总结翻译延伸研究方法的基础上,重点探讨了顺式调控元件(mRNA与新生肽链序列)对局部翻译延伸速率的调控作用,同时列举了翻译延伸调控对模板mRNA和蛋白质产物功能的影响,包括mRNA稳定性、蛋白质的合成与降解、蛋白质亚细胞定位以及蛋白质共翻译折叠等,以期吸引生命科学各领域的学者共同参与翻译延伸领域的研究。.

Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding.

Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.