bims-tricox Biomed News
on Translation, ribosomes and COX
Issue of 2023‒04‒30
six papers selected by
Yash Verma
University of Delhi South Campus
  1. The N-terminal region of yeast mitoribosomal Mrp7/bL27m protein serves to optimize translation of nascent chains competent for OXPHOS complex assembly.
  2. The Importance of Being RNA-est: considering RNA-mediated ribosome plasticity.
  3. Interplay between Inter-Subunit Rotation of the Ribosome and Binding of Translational GTPases.
  4. The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.
  5. A Correlation between 3'-UTR of OXA1 Gene and Yeast Mitochondrial Translation.
  6. eGFP as an All-in-One Tag for Purification of Membrane Proteins.
  1. FEBS Lett. 2023 Apr 28.
    The N-terminal region of yeast mitoribosomal Mrp7/bL27m protein serves to optimize translation of nascent chains competent for OXPHOS complex assembly.
    Jessica M Anderson, Jodie M Box, Rosemary A Stuart.
      The extreme N-terminal residues of the mitochondrial ribosomal bL27m proteins reside within the ribosomal peptidyl transferase center (PTC) and are conserved from their bacterial ancestors. Mutation or truncation of the N-terminal region of the yeast Mrp7/bL27m protein did not inhibit protein synthesis but significantly impacted the efficacy of the mitochondrial translational process with respect to yielding proteins competent to assemble into functional oxidative phosphorylation (OXPHOS) enzymes. The requirement for the N-terminal residues of Mrp7/bL27m to support normal mitotranslation was more apparent under respiratory growth. We demonstrate that the N-terminal region of Mrp7/bL27m impacts the environment of the PTC and speculate the bL27m proteins serve to fine-tune and optimize mitoribosomal activity with respect to the downstream fate of the nascent chain.
    DOI:  https://doi.org/10.1002/1873-3468.14631
  2. RNA Biol. 2023 Jan;20(1): 177-185
    The Importance of Being RNA-est: considering RNA-mediated ribosome plasticity.
    Christian Trahan, Marlene Oeffinger.
      For over 40 years, ribosomes were considered monolithic machines that translate the genetic code indiscriminately. However, over the past two decades, there have been a growing number of studies that suggest ribosomes to have a degree of compositional and functional adaptability in response to tissue type, cell environment and stimuli, cell cycle or development state. In such form, ribosomes themselves take an active part in translation regulation through an intrinsic adaptability provided by evolution, which furnished ribosomes with a dynamic plasticity that confers another layer of gene expression regulation. Yet despite the identification of various sources that give rise to ribosomal heterogeneity both at the protein and RNA level, its functional relevance is still debated, and many questions remain. Here, we will review aspects, including evolutionary ones, of ribosome heterogeneity emerging at the nucleic acid level, and aim to reframe ribosome 'heterogeneity' as an adaptive and dynamic process of plasticity.The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
    Keywords:  5.8S rRNA; heterogeneous ribosomes; rRNA evolution; rRNA expansion segments; rRNA heterogeneity; rRNA modifications; rRNA variants; ribosome plasticity
    DOI:  https://doi.org/10.1080/15476286.2023.2204581
  3. Int J Mol Sci. 2023 Apr 07. pii: 6878. [Epub ahead of print]24(8):
    Interplay between Inter-Subunit Rotation of the Ribosome and Binding of Translational GTPases.
    Ananya Das, Nichole Adiletta, Dmitri N Ermolenko.
      Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation.
    Keywords:  EF-G; inter-subunit rotation; ribosome; single-molecule Förster Resonance Energy Transfer (smFRET)
    DOI:  https://doi.org/10.3390/ijms24086878
  4. Life Sci Alliance. 2023 Jul;pii: e202201858. [Epub ahead of print]6(7):
    The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.
    Daniel Flores-Mireles, Yolanda Camacho-Villasana, Madhurya Lutikurti, Aldo E García-Guerrero, Guadalupe Lozano-Rosas, Victoria Chagoya, Emma Berta Gutiérrez-Cirlos, Ulrich Brandt, Alfredo Cabrera-Orefice, Xochitl Pérez-Martínez.
      Mitochondrial bc 1 complex from yeast has 10 subunits, but only cytochrome b (Cytb) subunit is encoded in the mitochondrial genome. Cytb has eight transmembrane helices containing two hemes b for electron transfer. Cbp3 and Cbp6 assist Cytb synthesis, and together with Cbp4 induce Cytb hemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cytb synthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Because Qcr7 resides near the Cytb carboxyl region, we wondered whether this region is important for Cytb synthesis/assembly. Although deletion of the Cytb C-region did not abrogate Cytb synthesis, the assembly-feedback regulation was lost, so Cytb synthesis was normal even if Qcr7 was missing. Mutants lacking the Cytb C-terminus were non-respiratory because of the absence of fully assembled bc 1 complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work, we demonstrate that the C-terminal region of Cytb is critical for regulation of Cytb synthesis and bc 1 complex assembly.
    DOI:  https://doi.org/10.26508/lsa.202201858
  5. J Fungi (Basel). 2023 Apr 05. pii: 445. [Epub ahead of print]9(4):
    A Correlation between 3'-UTR of OXA1 Gene and Yeast Mitochondrial Translation.
    Maryam Hajikarimlou, Mohsen Hooshyar, Noor Sunba, Nazila Nazemof, Mohamed Taha Moutaoufik, Sadhena Phanse, Kamaledin B Said, Mohan Babu, Martin Holcik, Bahram Samanfar, Myron Smith, Ashkan Golshani.
      Mitochondria possess their own DNA (mtDNA) and are capable of carrying out their transcription and translation. Although protein synthesis can take place in mitochondria, the majority of the proteins in mitochondria have nuclear origin. 3' and 5' untranslated regions of mRNAs (3'-UTR and 5'-UTR, respectively) are thought to play key roles in directing and regulating the activity of mitochondria mRNAs. Here we investigate the association between the presence of 3'-UTR from OXA1 gene on a prokaryotic reporter mRNA and mitochondrial translation in yeast. OXA1 is a nuclear gene that codes for mitochondrial inner membrane insertion protein and its 3'-UTR is shown to direct its mRNA toward mitochondria. It is not clear, however, if this mRNA may also be translated by mitochondria. In the current study, using a β-galactosidase reporter gene, we provide genetic evidence for a correlation between the presence of 3'-UTR of OXA1 on an mRNA and mitochondrial translation in yeast.
    Keywords:  gene expression; messenger RNA; mitochondria; translation; untranslated regions; yeast
    DOI:  https://doi.org/10.3390/jof9040445
  6. Methods Mol Biol. 2023 ;2652 171-186
    eGFP as an All-in-One Tag for Purification of Membrane Proteins.
    Tomáš Heger, Charlott Stock, Michelle Juknaviciute Laursen, Michael Habeck, Thibaud Dieudonné, Poul Nissen.
      Within the last decade, cryo-electron microscopy has revolutionized our understanding of membrane proteins, but they still represent challenging targets for biochemical and structural studies. The first obstacle is often to obtain high production levels of correctly folded target protein. In these cases, the use of eGFP tags is an efficient strategy, as it allows rapid screenings of expression systems, constructs, and detergents for solubilization. Additionally, eGFP tags can now be used for affinity purification with recently developed nanobodies. Here we present a series of methods based on enhanced green fluorescent protein (eGFP) fluorescence to efficiently screen for production and stabilization of detergent-solubilized eGFP-tagged membrane proteins produced in S. cerevisiae via in-gel fluorescence SDS-PAGE and fluorescence-detection size-exclusion chromatography (FSEC). Additionally, we present a protocol describing the production of affinity resin based on eGFP-binding nanobodies produced in E. coli. We showcase the purification of human ATP7B, a copper transporting P-type ATPase, as an example of the applicability of the methods.
    Keywords:  ATP7B; FSEC; In-gel fluorescence; Membrane protein; Nanobody; Purification; eGFP
    DOI:  https://doi.org/10.1007/978-1-0716-3147-8_9
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