The ABCF proteins inEscherichia coliindividually alleviate nascent peptide-induced noncanonical translations

Abstract

Organisms possess a wide variety of proteins with a diverse repertoire of amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the translation elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors, enabling the synthesis of diverse proteins.In this study, we have revealed thatEscherichia coliATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various noncanonical translations induced by nascent peptide sequences within the exit tunnel of the ribosome. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent ribosome stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that the ATP hydrolysis-coupled structural rearrangement and interdomain linker sequence between the two nucleotide-binding domains play crucial roles in alleviating the noncanonical translations. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences.Significance statementProteins, that constitute living organisms, exhibit a diverse range of amino acid sequences. However, it has become evident that ribosomes have difficulties in synthesizing certain amino acid sequences, including the poly-basic, poly-acidic, and poly-proline sequences. The mechanisms underlying the expression of proteins with such challenging sequences remain largely elusive. In this study, we have unveiled that translation factor ABCF proteins inEscherichia colipromote various kinds of problematic amino acid sequences that inhibit efficient translation. Through the actions of translation elongation factors including the ABCF proteins, the translation system acquires robustness in synthesizing a vast repertoire of amino acid sequences.

YfmR is a translation factor that prevents ribosome stalling and cell death in the absence of EF-P

“…However, it is possible that, like EF-P, Uup does not stimulate peptide bond formation between fMet-Phe, but may stimulate peptide bond formation between fMet and other amino acids (26, 29). Indeed, a recent preprint showed that Uup increased production of a peptide encoding a polyproline tract in vitro (67). Moreover, Uup-EQ2 has similar binding affinity for both initiating and elongating ribosomes (54), further suggesting that Uup may function in elongation.…”

Global regulation via modulation of ribosome pausing by the ABC-F protein EttA

“…Firstly, in the case of aceB , the absence of a toeprint signal in the absence of EttA, juxtaposed with its presence in the presence of His 6 -EttA-EQ 2 , strongly suggests ribosome dissociation that can be stabilized by EttA-EQ 2 . This possible dissociation is further corroborated by a recent preprint in which the authors show that the introduction of three Glu residues at the beginning of the LacZ protein sequence triggers ribosome dissociation during in vitro synthesis, as evidenced by the formation of a peptidyl-tRNA, which can be rescued by EttA 58 . Secondly, for yjbJ , a distinct ribosome stalling event is observed, characterized by the detectable toeprinting signal.…”

“…Ribosomal dissociation during elongation of acidic-residues has been previously demonstrated for MgtL and other proteins in prokaryotes 56,57 and eukaryotes 67 , this effect was described as Intrinsic Ribosome Destabilization (IRD). Moreover, a recent report suggest that EttA can prevent the IRD in vitro 55 .…”

“…Ribosomal dissociation during elongation of acidic-residues has been previously demonstrated for MgtL and other proteins in prokaryotes 56,57 and eukaryotes 67 , this effect was described as Intrinsic Ribosome Destabilization (IRD). Moreover, a recent report suggest that EttA can prevent the IRD in vitro 55 . This model draws parallels with the function of the translation factor EF-P 68 , which enhances translation of polyproline-containing proteins 24,25 .…”

EF-P and its paralog EfpL (YeiP) differentially control translation of proline-containing sequences

“…The importance to alleviate ribosome stalling at consecutive prolines is further underlined by the existence of additional rescue systems namely the A TP- B inding C assette family- F (ABCF) protein Uup in E. coli and its ortholog YfmR in B. subtilis 37,59–61 . In interplay with EF-P, Uup/YfmR and EfpL can facilitate translation of XP(P)X containing proteins.…”