bims-mitran 2021-08-22 papers

bims-mitran

Biomed News

on Mitochondrial translation

Issue of 2021–08–22
eight papers selected by
Andreas Kohler, Stockholm University

IRC3 regulates mitochondrial translation in response to metabolic cues in Saccharomyces cerevisiae.
Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.
Comparative Analysis of Structural and Dynamical Features of Ribosome Upon Association With mRNA Reveals Potential Role of Ribosomal Proteins.
Twice exploration of tRNA +1 frameshifting in an elongation cycle of protein synthesis.
Charcot-Marie-Tooth mutation in glycyl-tRNA synthetase stalls ribosomes in a pre-accommodation state and activates integrated stress response.
Codon usage and protein length-dependent feedback from translation elongation regulates translation initiation and elongation speed.
Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation.
Mechanisms that ensure speed and fidelity in eukaryotic translation termination.

Mol Cell Biol. 2021 Aug 16. MCB0023321

IRC3 regulates mitochondrial translation in response to metabolic cues in Saccharomyces cerevisiae.

Jaswinder Kaur, Kaustuv Datta.

Mitochondrial oxidative phosphorylation (OXPHOS) enzymes are made up of dual genetic origin. Mechanisms regulating the expression of nuclear-encoded OXPHOS subunits in response to metabolic cues (glucose vs. glycerol), is significantly understood while regulation of mitochondrially encoded OXPHOS subunits is poorly defined. Here, we show that IRC3 a DEAD/H box helicase, previously implicated in mitochondrial DNA maintenance, is central to integrating metabolic cues with mitochondrial translation. Irc3 associates with mitochondrial small ribosomal subunit in cells consistent with its role in regulating translation elongation based on Arg8m reporter system. IRC3 deleted cells retained mitochondrial DNA despite growth defect on glycerol plates. Glucose grown Δirc3ρ+ and irc3 temperature-sensitive cells at 370C have reduced translation rates from majority of mRNAs. In contrast, when galactose was the carbon source, reduction in mitochondrial translation was observed predominantly from Cox1 mRNA in Δirc3ρ+ but no defect was observed in irc3 temperature-sensitive cells, at 370C. In support, of a model whereby IRC3 responds to metabolic cues to regulate mitochondrial translation, suppressors of Δirc3 isolated for restoration of growth on glycerol media restore mitochondrial protein synthesis differentially in presence of glucose vs. glycerol.

DOI: https://doi.org/10.1128/MCB.00233-21
Nucleic Acids Res. 2021 Aug 17. pii: gkab665. [Epub ahead of print]

Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.

Ting Su, Renuka Kudva, Thomas Becker, Robert Buschauer, Tobias Komar, Otto Berninghausen, Gunnar von Heijne, Jingdong Cheng, Roland Beckmann.

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

DOI: https://doi.org/10.1093/nar/gkab665
Front Mol Biosci. 2021 ;8 654164

Comparative Analysis of Structural and Dynamical Features of Ribosome Upon Association With mRNA Reveals Potential Role of Ribosomal Proteins.

Sneha Bheemireddy, Sankaran Sandhya, Narayanaswamy Srinivasan.

  Ribosomes play a critical role in maintaining cellular proteostasis. The binding of messenger RNA (mRNA) to the ribosome regulates kinetics of protein synthesis. To generate an understanding of the structural, mechanistic, and dynamical features of mRNA recognition in the ribosome, we have analysed mRNA-protein interactions through a structural comparison of the ribosomal complex in the presence and absence of mRNA. To do so, we compared the 3-Dimensional (3D) structures of components of the two assembly structures and analysed their structural differences because of mRNA binding, using elastic network models and structural network-based analysis. We observe that the head region of 30S ribosomal subunit undergoes structural displacement and subunit rearrangement to accommodate incoming mRNA. We find that these changes are observed in proteins that lie far from the mRNA-protein interface, implying allostery. Further, through perturbation response scanning, we show that the proteins S13, S19, and S20 act as universal sensors that are sensitive to changes in the inter protein network, upon binding of 30S complex with mRNA and other initiation factors. Our study highlights the significance of mRNA binding in the ribosome complex and identifies putative allosteric sites corresponding to alterations in structure and/or dynamics, in regions away from mRNA binding sites in the complex. Overall, our work provides fresh insights into mRNA association with the ribosome, highlighting changes in the interactions and dynamics of the ribosome assembly because of the binding.

Keywords:  allostery; dynamics; flexibility; messenger RNA (mRNA); normal mode analysis; ribosome

DOI:  https://doi.org/10.3389/fmolb.2021.654164
Nucleic Acids Res. 2021 Aug 20. pii: gkab734. [Epub ahead of print]

Twice exploration of tRNA +1 frameshifting in an elongation cycle of protein synthesis.

Howard Gamper, Yujia Mao, Isao Masuda, Henri McGuigan, Gregor Blaha, Yuhong Wang, Shoujun Xu, Ya-Ming Hou.

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with -1 frameshifting.

DOI: https://doi.org/10.1093/nar/gkab734
Nucleic Acids Res. 2021 Aug 17. pii: gkab730. [Epub ahead of print]

Charcot-Marie-Tooth mutation in glycyl-tRNA synthetase stalls ribosomes in a pre-accommodation state and activates integrated stress response.

Samantha Mendonsa, Nicolai von Kuegelgen, Lucija Bujanic, Marina Chekulaeva.

Toxic gain-of-function mutations in aminoacyl-tRNA synthetases cause a degeneration of peripheral motor and sensory axons, known as Charcot-Marie-Tooth (CMT) disease. While these mutations do not disrupt overall aminoacylation activity, they interfere with translation via an unknown mechanism. Here, we dissect the mechanism of function of CMT mutant glycyl-tRNA synthetase (CMT-GARS), using high-resolution ribosome profiling and reporter assays. We find that CMT-GARS mutants deplete the pool of glycyl-tRNAGly available for translation and inhibit the first stage of elongation, the accommodation of glycyl-tRNA into the ribosomal A-site, which causes ribosomes to pause at glycine codons. Moreover, ribosome pausing activates a secondary repression mechanism at the level of translation initiation, by inducing the phosphorylation of the alpha subunit of eIF2 and the integrated stress response. Thus, CMT-GARS mutant triggers translational repression via two interconnected mechanisms, affecting both elongation and initiation of translation.

DOI: https://doi.org/10.1093/nar/gkab730
Nucleic Acids Res. 2021 Aug 20. pii: gkab729. [Epub ahead of print]

Codon usage and protein length-dependent feedback from translation elongation regulates translation initiation and elongation speed.

Xueliang Lyu, Qian Yang, Fangzhou Zhao, Yi Liu.

Essential cellular functions require efficient production of many large proteins but synthesis of large proteins encounters many obstacles in cells. Translational control is mostly known to be regulated at the initiation step. Whether translation elongation process can feedback to regulate initiation efficiency is unclear. Codon usage bias, a universal feature of all genomes, plays an important role in determining gene expression levels. Here, we discovered that there is a conserved but codon usage-dependent genome-wide negative correlation between protein abundance and CDS length. The codon usage effects on protein expression and ribosome flux on mRNAs are influenced by CDS length; optimal codon usage preferentially promotes production of large proteins. Translation of mRNAs with long CDS and non-optimal codon usage preferentially induces phosphorylation of initiation factor eIF2α, which inhibits translation initiation efficiency. Deletion of the eIF2α kinase CPC-3 (GCN2 homolog) in Neurospora preferentially up-regulates large proteins encoded by non-optimal codons. Surprisingly, CPC-3 also inhibits translation elongation rate in a codon usage and CDS length-dependent manner, resulting in slow elongation rates for long CDS mRNAs. Together, these results revealed a codon usage and CDS length-dependent feedback mechanism from translation elongation to regulate both translation initiation and elongation kinetics.

DOI: https://doi.org/10.1093/nar/gkab729
J Am Chem Soc. 2021 Aug 17.

Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation.

Yumi Koga, Eileen M Hoang, Yongho Park, Alexander F A Keszei, Jason Murray, Sichen Shao, Brian B Liau.

Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX's extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX (8) occupies the same site as CHX, competing with the 3' end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

DOI: https://doi.org/10.1021/jacs.1c05146
Science. 2021 08 20. 373(6557): 876-882

Mechanisms that ensure speed and fidelity in eukaryotic translation termination.

Michael R Lawson, Laura N Lessen, Jinfan Wang, Arjun Prabhakar, Nicholas C Corsepius, Rachel Green, Joseph D Puglisi.

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bound together to recognize stop codons rapidly and elicit termination through a tightly regulated, multistep process that resembles transfer RNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

DOI: https://doi.org/10.1126/science.abi7801