bims-tricox Biomed News
on Translation, ribosomes and COX
Issue of 2022‒07‒31
nine papers selected by
Yash Verma
University of Delhi South Campus
  1. Uncovering translation roadblocks during the development of a synthetic tRNA.
  2. Increasing Complexity of Ribosomes and Their Biogenesis.
  3. Precise tuning of bacterial translation initiation by non-equilibrium 5'-UTR unfolding observed in single mRNAs.
  4. The future of yeast research.
  5. mRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation.
  6. Large protein complex interfaces have evolved to promote cotranslational assembly.
  7. Catalytic cycling of human mitochondrial Lon protease.
  8. A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins.
  9. AlphaFold predicts the most complex protein knot and composite protein knots.
  1. Nucleic Acids Res. 2022 Jul 27. pii: gkac576. [Epub ahead of print]
    Uncovering translation roadblocks during the development of a synthetic tRNA.
    Arjun Prabhakar, Natalie Krahn, Jingji Zhang, Oscar Vargas-Rodriguez, Miri Krupkin, Ziao Fu, Francisco J Acosta-Reyes, Xueliang Ge, Junhong Choi, Ana Crnković, Måns Ehrenberg, Elisabetta Viani Puglisi, Dieter Söll, Joseph Puglisi.
      Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
    DOI:  https://doi.org/10.1093/nar/gkac576
  2. Int J Mol Sci. 2022 Jul 27. pii: 8264. [Epub ahead of print]23(15):
    Increasing Complexity of Ribosomes and Their Biogenesis.
    Lasse Lindahl.
      According to the classic ribosome model, developed in the 1960s and 1970s, its only function is to translate the four-letter nucleic acid code into the 20 amino acid peptide-code, while polymerizing amino acids into peptides with the help of a large complement of tRNAs and translation factors that cycle on and off the ribosome [...].
    DOI:  https://doi.org/10.3390/ijms23158264
  3. Nucleic Acids Res. 2022 Jul 27. pii: gkac635. [Epub ahead of print]
    Precise tuning of bacterial translation initiation by non-equilibrium 5'-UTR unfolding observed in single mRNAs.
    Sujay Ray, Shiba S Dandpat, Surajit Chatterjee, Nils G Walter.
      Noncoding, structured 5'-untranslated regions (5'-UTRs) of bacterial messenger RNAs (mRNAs) can control translation efficiency by forming structures that either recruit or repel the ribosome. Here we exploit a 5'-UTR embedded preQ1-sensing, pseudoknotted translational riboswitch to probe how binding of a small ligand controls recruitment of the bacterial ribosome to the partially overlapping Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5'-UTR during mRNA accommodation into the mRNA binding cleft. Ligand binding to the riboswitch stabilizes the structure to both antagonize 30S recruitment and accelerate 30S dissociation. Proximity of the 5'-UTR and stability of the SD:anti-SD interaction both play important roles in modulating the initial 30S-mRNA interaction. Finally, depletion of small ribosomal subunit protein S1, known to help resolve structured 5'-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5'-UTR during accommodation provides a mechanistic understanding of how translation efficiency is governed by riboswitches and other dynamic structure motifs embedded upstream of the translation initiation site of bacterial mRNAs.
    DOI:  https://doi.org/10.1093/nar/gkac635
  4. Microbiology (Reading). 2022 Jul;168(7):
    The future of yeast research.
    Stephen G Oliver.
      
    DOI:  https://doi.org/10.1099/mic.0.001233
  5. Nucleic Acids Res. 2022 Jul 25. pii: gkac631. [Epub ahead of print]
    mRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation.
    Burak Çetin, Seán E O'Leary.
      mRNA 5' cap recognition by eIF4F is a key element of eukaryotic translational control. Kinetic differences in eIF4F-mRNA interactions have long been proposed to mediate translation-efficiency differences between mRNAs, and recent transcriptome-wide studies have revealed significant heterogeneity in eIF4F engagement with differentially-translated mRNAs. However, detailed kinetic information exists only for eIF4F interactions with short model RNAs. We developed and applied single-molecule fluorescence approaches to directly observe real-time Saccharomyces cerevisiae eIF4F subunit interactions with full-length polyadenylated mRNAs. We found that eIF4E-mRNA association rates linearly anticorrelate with mRNA length. eIF4G-mRNA interaction accelerates eIF4E-mRNA association in proportion to mRNA length, as does an eIF4F-independent activity of eIF4A, though cap-proximal secondary structure still plays an important role in defining the final association rates. eIF4F-mRNA interactions remained dominated by effects of eIF4G, but were modulated to different extents for different mRNAs by the presence of eIF4A and ATP. We also found that eIF4A-catalyzed ATP hydrolysis ejects eIF4E, and likely eIF4E•eIF4G from the mRNA after initial eIF4F•mRNA complex formation, suggesting a mechanism to prepare the mRNA 5' end for ribosome recruitment. Our results support a role for mRNA-specific, factor-driven eIF4F association rates in kinetically controlling translation.
    DOI:  https://doi.org/10.1093/nar/gkac631
  6. Elife. 2022 Jul 28. pii: e79602. [Epub ahead of print]11
    Large protein complex interfaces have evolved to promote cotranslational assembly.
    Mihaly Badonyi, Joseph A Marsh.
      Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this efficiency is by seeding assembly pathways during translation via the cotranslational assembly of subunits. While recent evidence suggests that such cotranslational assembly is widespread, little is known about the properties of protein complexes associated with the phenomenon. Here, using a combination of proteome-specific protein complex structures and publicly available ribosome profiling data, we show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, we compared the sizes of first and last translated interfaces of heteromeric subunits in bacterial, yeast, and human complexes. When considering all together, we observe the N-terminal interface to be larger than the C-terminal interface 54% of the time, increasing to 64% when we exclude subunits with only small interfaces, which are unlikely to cotranslationally assemble. This strongly suggests that large interfaces have evolved as a means to maximise the chance of successful cotranslational subunit binding.
    Keywords:  E. coli; S. cerevisiae; computational biology; evolutionary biology; human; systems biology
    DOI:  https://doi.org/10.7554/eLife.79602
  7. Structure. 2022 Jul 12. pii: S0969-2126(22)00269-6. [Epub ahead of print]
    Catalytic cycling of human mitochondrial Lon protease.
    Inayathulla Mohammed, Kai A Schmitz, Niko Schenck, Dimitrios Balasopoulos, Annika Topitsch, Timm Maier, Jan Pieter Abrahams.
      The mitochondrial Lon protease (LonP1) regulates mitochondrial health by removing redundant proteins from the mitochondrial matrix. We determined LonP1 in eight nucleotide-dependent conformational states by cryoelectron microscopy (cryo-EM). The flexible assembly of N-terminal domains had 3-fold symmetry, and its orientation depended on the conformational state. We show that a conserved structural motif around T803 with a high similarity to the trypsin catalytic triad is essential for proteolysis. We show that LonP1 is not regulated by redox potential, despite the presence of two conserved cysteines at disulfide-bonding distance in its unfoldase core. Our data indicate how sequential ATP hydrolysis controls substrate protein translocation in a 6-fold binding change mechanism. Substrate protein translocation, rather than ATP hydrolysis, is a rate-limiting step, suggesting that LonP1 is a Brownian ratchet with ATP hydrolysis preventing translocation reversal. 3-fold rocking motions of the flexible N-domain assembly may assist thermal unfolding of the substrate protein.
    Keywords:  AAA+ protein; chaperone; molecular motor; proteolysis
    DOI:  https://doi.org/10.1016/j.str.2022.06.006
  8. Elife. 2022 Jul 25. pii: e77706. [Epub ahead of print]11
    A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins.
    Layla Drwesh, Benjamin Heim, Max Graf, Linda Kehr, Lea Hansen-Palmus, Mirita Franz-Wachtel, Boris Macek, Hubert Kalbacher, Johannes Buchner, Doron Rapaport.
      Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.
    Keywords:  S. cerevisiae; biochemistry; chemical biology
    DOI:  https://doi.org/10.7554/eLife.77706
  9. Protein Sci. 2022 Aug;31(8): e4380
    AlphaFold predicts the most complex protein knot and composite protein knots.
    Maarten A Brems, Robert Runkel, Todd O Yeates, Peter Virnau.
      The computer artificial intelligence system AlphaFold has recently predicted previously unknown three-dimensional structures of thousands of proteins. Focusing on the subset with high-confidence scores, we algorithmically analyze these predictions for cases where the protein backbone exhibits rare topological complexity, that is, knotting. Amongst others, we discovered a 71 -knot, the most topologically complex knot ever found in a protein, as well several six-crossing composite knots comprised of two methyltransferase or carbonic anhydrase domains, each containing a simple trefoil knot. These deeply embedded composite knots occur evidently by gene duplication and interconnection of knotted dimers. Finally, we report two new five-crossing knots including the first 51 -knot. Our list of analyzed structures forms the basis for future experimental studies to confirm these novel-knotted topologies and to explore their complex folding mechanisms.
    Keywords:  AlphaFold; composite knots; protein knots; protein topology
    DOI:  https://doi.org/10.1002/pro.4380
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