This work structurally characterize chimeric ribosomes derived from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae using cryo-electron microscopy and uncover a potential relationship between 16S ribosomal RNA (rRNA) stability and translation efficiency, providing new insights into rRNA structural malleability.
Abstract
Abstract The ribosomal RNA sequence governs translation dynamics, yet understanding how changes beyond the conserved catalytic centers influence kinetics and protein yield remains limited. Using orthogonal ribosome phage-assisted continuous evolution, we recently reported chimeric ribosomes derived from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae endowed with elevated orthogonal translation activity as compared to their starting counterparts. Here, we structurally characterize these kinetically enhanced ribosomes using cryo-electron microscopy and uncover a potential relationship between 16S ribosomal RNA (rRNA) stability and translation efficiency. Compared to their naive starting points, evolved ribosomes exhibit extensive RNA structural adaptation, often introduced by mismatches at key helical junctions, which leads to local RNA-protein rearrangements and destabilizes non-canonical base pairs. Compensatory mutations that restore base-pairing stability and eliminate flexibility reduced translational activity to wild-type levels. Across trajectories, increased translational output correlates with subtle, localized changes in the 16S rRNA sequence that introduce limited structural destabilization at specific elements. Taken together, our work provides new insights into rRNA structural malleability and establishes principles for engineering ribosomes with altered translation properties.
It is proposed that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts have evolved structural features that fine-tune protein levels that may be prevalent in bacteria which exhibit uncoupled transcription and translation.
Zachory M. Park, Christina R. Savage, Amanda R. Decker-Farrell et al.· Cell Reports· 0 citations
Abstract Ribosomal protein L41 (RPL41 or eL41) is the smallest ribosomal protein and forms the eukaryote-specific bridge, eB14, near the decoding center; however, its role in mammalian translation remains unclear. In this study, we established RPL41-deficient models of human HEK293T cells and mice to define its function. Cryo-electron microscopy revealed that RPL41 constrains intersubunit conformational dynamics without inducing major local static rearrangements. Loss of RPL41 altered A-site dynamics, slowed elongation, modestly increased amino acid misincorporation, and modestly enhanced readthrough of collision-inducing reporter sequences. Quantitative proteomic analysis suggested that these translational defects compromise long-protein homeostasis, as evidenced by increased insolubility and reduced abundance of long proteins. In vivo, Rpl41−/− mice were viable but exhibited growth retardation and decreased abundance of long proteins in tissues. Our findings reveal a conserved role for RPL41 in maintaining ribosome dynamics and translational fidelity, indicating that RPL41 supports ribosome function and long-protein homeostasis in mammals.
Mina Hirata, Maho Fujino, Kazuya Ichihara et al.· Nucleic Acids Research· 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
Accurate termination of protein synthesis is paramount for the integrity of the cellular proteome, yet the dynamics and fidelity of ribosome termination remain poorly understood. Here, we establish a profiling strategy to capture terminating ribosomes in mammalian cells and reveal a substantial heterogeneity in ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that promotes termination pausing, a finding supported by massively parallel reporter assays. Unexpectedly, reduced termination pausing increases the likelihood of stop codon slippage, giving rise to proteins with heterogeneous C-terminal extensions. Mechanistically, we show that sequence-dependent termination pausing is consistent with post-decoding mRNA scanning by the 3′ end of 18 S rRNA. We further uncover tissue-specific patterns of termination pausing that correlate with the stoichiometry of Rps26, which potentially modulates mRNA:rRNA interactions. Together, these results suggest termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.
Accurate termination of protein synthesis is paramount for the integrity of cellular proteome, yet the dynamics and fidelity of ribosome termination remain poorly understood. Here, we establish a profiling strategy to capture terminating ribosomes in mammalian cells and reveal a substantial heterogeneity in ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that promotes termination pausing, a finding validated by massively paralleled reporter assays. Unexpectedly, reduced termination pausing increases the likelihood of stop codon slippage, giving rise to proteins with heterogenous C-terminal extensions. Mechanistically, we show that sequence-dependent termination pausing arises from post-decoding mRNA scanning by the 3’ end of 18S rRNA. We further uncover tissue-specific patterns of termination pausing that correlates with the stoichiometry of Rps26, which modulates mRNA:rRNA interactions. Together, these results establish termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.