Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

Abstract

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.translation pausing | nascent polypeptides j Escherichia coli | peptidyl-tRNA

Quality over quantity: optimizing co-translational protein folding with non-‘optimal’ synonymous codons

“…More broadly, increasing the expression of low abundance tRNAs (which often correlate with rare codons) increased the aggregation of many E. coli cytoplasmic proteins [36], suggesting that slower translation might lead to higher folding yields for a variety of proteins. Consistent with this hypothesis, a correlation has been observed between translational pauses in E. coli coding sequences and protein solubility [37]…”

Depletion of Shine-Dalgarno Sequences Within Bacterial Coding Regions Is Expression Dependent

“…Indeed, the existence of SD-mediated pauses has not been confirmed using several other experimental methods (Borg and Ehrenberg 2015; Chadani et al 2016; Mohammad et al 2016). Thus, remains unclear what role, if any, SD sequences within protein coding genes have in modulating translation speed (Figure 1B).…”

Strolling Toward New Concepts

“…Polypeptides of less than full length do not necessarily represent nascent states of the proteins because they can arise for various reasons, including proteolysis. The fact that nascent polypeptides carry a tRNA at the growing end can be exploited for the valid detection of cellular nascent polypeptides, by either two-dimensional (before and after tRNA removal; see above section A TCA Biologist; Figure 2) or one-dimensional (searching for RNase-sensitive materials) electrophoresis (16,44). Using such a method, I collaborated with Tatsuhiko Abo and Yuhei Chadani to show that E. coli cells can produce significant fractions of futile translation products in the forms of polypeptidyl-tRNAs, although these are normally prevented from appearing by the ribosome-rescuing pathways (44).…”

“…Previously, such systems for quality control of translation complexes had only been studied using model nonstop constructs. Yuhei later joined my lab and profiled nascent chains of individually expressed E. coli genes; he found that translational pausing, occurring in different modes, is unexpectedly widespread (16). Although the ribosome-profiling technique has higher throughput and resolution, it is important to detect and characterize translational pausing more directly using methods such as ours.…”