Aug 2026· Microbiology and Molecular Biology Reviews· 0 citations· 173 references
Medicine
TL;DR
Recently identified translation factors that prevent ribosome stalling at polyproline tracts in bacteria are reviewed.
Abstract
SUMMARY Ribosomes produce the staggering array of proteins that perform the structural and enzymatic feats of the cell. Therefore, most of the cell’s energy goes toward producing ribosomes and the work performed by them. The work of the ribosome is relatively simple—decode mRNA codons and catalyze the formation of peptide bonds. By marching iteratively along the length of an open reading frame, a complete peptide is produced. Ribosomes catalyze the formation of peptide bonds at a rate of approximately 15 amino acids per second. However, bonds between amino acids do not form with equal efficiency, and ribosomes can become stalled at difficult-to-translate sequences. Proline is unique among the amino acids in that its side chain is covalently bonded to the peptide backbone to form a rigid ring. The rigidity of proline, and especially tracts of proline, makes it a difficult substrate for peptide bond formation, but it is also an essential motif in many protein structures. Elongation factor P (EF-P) is the star player for facilitating translation of polyproline tracts. However, recently identified factors play an important supporting role, and loss of these factors incurs a severe fitness defect in the absence of EF-P. These factors include an EF-P paralog, EfpL, as well as the ABCF ATPase YfmR/Uup and YebC2. The abundance and partial redundancy of factors that prevent ribosome stalling at polyprolines highlights the structural importance of polyproline tracts and the need to facilitate their translation. Here, we review recently identified translation factors that prevent ribosome stalling at polyprolines in bacteria.
The recognition mechanism of Kozak mRNA, typically comprising purines in the - 3 and +4 positions flanking the AUG start codon, has remained enigmatic for decades. To address this fundamental function in eukaryotes during translation initiation, we analysed the cryo-EM structures of human 48S preinitiation complexes with mRNA point mutations. They reveal a fan-like intercalation of the pre-codon triplet into the 18S ribosomal RNA (rRNA), while the -3 purine as opposed to the pyrimidine in the non-Kozak context favours stabilization of the ternary complex between initiation factor eIF2 and initiator tRNA. Specificity towards the +4 purine is achieved beyond a single residue recognition by mutual conformational adaptations of eIF1A, mRNA and rRNA and involves the insertion of a reading head in which decoding residue A1825 (rRNA) stacks with the A-site codon to stabilize the fully accommodated state. Hence, instead of relying on base pairing as in bacteria, the specific recognition of the Kozak sequence on eukaryotic ribosomes is based on an induced-fit mechanism that triggers a conformational readout of the mRNA.
Ottilie von Loeffelholz, Charles Barchet, Samuel Holvec et al.· bioRxiv· 2 citations
The ribosome’s DNA-encoded production of defined polymer sequences is naturally limited to 22 amino acids. Although the translation machinery has the latent capacity to polymerize backbone-modified substrates, including β-amino acids, this potential is constrained by the intrinsic α-selectivity of native aminoacyl-tRNA synthetases. Here, we address this limitation by “reverse engineering” the Escherichia coli protein ligase EpmA. Naturally activating (R)-β-lysine, EpmA evolved to discard its tRNA-binding domain in favor of protein recognition. By grafting the anticodon-binding domain of the canonical lysyl-tRNA synthetase, LysRS, onto EpmA, we created the chimeric enzyme chEpmA. To our knowledge, this represents the first successful reprogramming of a protein ligase into a functional aminoacyl-tRNA synthetase. We demonstrate that chEpmA serves as a versatile dual-specificity platform: it efficiently charges tRNAs with the non-canonical backbone (R)-β-lysine, and a single substitution unlocks the scaffold for α-substrates, thereby enabling a broad spectrum of post-translational modifications previously inaccessible to genetic code expansion. This repertoire ranges from acylated lysines such as Nε-succinyl-(S)-α lysine (Ksucc) and bulky modifications such as biocytin to advanced glycation end products (AGEs) including Nε-carboxymethyl-(S)-α lysine (CML). Our work establishes a structural blueprint for mobilizing non-canonical substrates, paving the way for the biosynthesis of protease-resistant peptidomimetics and next-generation therapeutics.
Giovanni Gallo, Alina Sieber, Michael Hellwig et al.· bioRxiv· 0 citations
Translating ribosomes can stall on mRNA for various reasons, including nuclease cleavage, arrest peptide sequences or ribosome collisions. In Escherichia coli, several ribosome rescue factors act to release ribosomes stalled at the 3' end of mRNA. Among these factors, ArfB can rescue stalled ribosomes without the help of other factors. ArfB consists of an N-terminal domain containing the catalytic GGQ motif, which mediates peptidyl-tRNA hydrolysis, and a C-terminal extension that functions as a sensor for recognizing stalled ribosomes. However, how these two regions coordinate to resolve ribosome stalling remains unclear. Here, using a reconstituted translation system, we found the functional importance of histidine residues in the N-terminal domain of ArfB. In particular, histidine at position 62 in E. coli ArfB was required for stop codon-independent fMet-tRNA hydrolysis, whereas its substitution did not affect affinity for the ribosome. Furthermore, directed hydroxyl radical probing revealed that H62A mutation did not significantly alter the overall positioning of either the N-terminal domain or the C-terminal extension of ArfB on the ribosome. These findings suggest that H62 contributes to ArfB function during a step following initial ribosome binding, thereby facilitating peptidyl-tRNA hydrolysis.
Daisuke Kurita, Kotone Furusawa, Takumi Kasuya et al.· RNA: A publication of the RN...· 0 citations
It is concluded that the early emergence of the Rossmann fold reflects the chemical and physical constraints of protein folding, explaining both its profound antiquity and sustained longevity.
Koh Seya, Tatsuya Corlett, Hamza Giaffar et al.· bioRxiv· 0 citations
Trans-translation and bL27 data are linked and support a model in which the amino terminus of bL27 acts as a gatekeeper to prevent tmRNA from sterically interfering with tRNA (transfer RNA) on the ribosome.
Divyasorubini Seerpatham, George Wanes, Chathuri Pathirage et al.· Science Advances· 0 citations
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