bims-tricox 2022-07-03 papers

bims-tricox

Biomed News

on Translation, ribosomes and COX

Issue of 2022–07–03
ten papers selected by
Yash Verma, University of Delhi South Campus

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine.
Evidence for selection on SARS-CoV-2 RNA translation revealed by the evolutionary dynamics of mutations in UTRs and CDSs.
Characterizing the Electron Transport Chain: Structural Approach.
Unraveling docking and initiation of mRNA export through the nuclear pore complex.
In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.
Vfold-Pipeline: a web server for RNA 3D structure prediction from sequences.
Membrane Protein Production in Escherichia coli: Protocols and Rules.
Signal Recognition Particle in Human Diseases.
How does mitochondrial function relate to thermogenic capacity and basal metabolic rate in small birds?
Pervasive translation of circular RNAs driven by short IRES-like elements.

Nucleic Acids Res. 2022 Jun 29. pii: gkac548. [Epub ahead of print]

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine.

Egor A Syroegin, Elena V Aleksandrova, Yury S Polikanov.

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

DOI: https://doi.org/10.1093/nar/gkac548
RNA Biol. 2022 Jan;19(1): 866-876

Evidence for selection on SARS-CoV-2 RNA translation revealed by the evolutionary dynamics of mutations in UTRs and CDSs.

Lin Zhu, Qi Wang, Weiyu Zhang, Hao Hu, Kexin Xu.

  RNA translation is the rate-limiting step when cells synthesize proteins. Elevating translation efficiency (TE) is intuitively beneficial. Particularly, when viruses invade host cells, how to compete with endogenous RNAs for efficient translation is a major issue to be resolved. We collected millions of worldwide SARS-CoV-2 sequences during the past year and traced the dynamics of allele frequency of every mutation. We defined adaptive and deleterious mutations according to the rise and fall of their frequencies along time. For 5'UTR and synonymous mutations in SARS-CoV-2, the selection on TE is evident near start codons. Adaptive mutations generally decrease GC content while deleterious mutations increase GC content. This trend fades away with increasing distance to start codons. Mutations decreasing GC content near start codons would unravel the complex RNA structure and facilitate translation initiation, which are beneficial to SARS-CoV-2, and vice versa. During this evolutionary arms race between human and virus, SARS-CoV-2 tries to improve its cis elements to compete with host RNAs for rapid translation.

Keywords:  RNA structure; SARS-CoV-2; allele frequency; mutation; translation

DOI:  https://doi.org/10.1080/15476286.2022.2092351
Methods Mol Biol. 2022 ;2497 107-115

Characterizing the Electron Transport Chain: Structural Approach.

Ting Liang, Janice Deng, Bijaya Nayak, Xin Zou, Yuji Ikeno, Yidong Bai.

  The mitochondrial respiratory chain which carries out the oxidative phosphorylation (OXPHOS) consists of five multi-subunit protein complexes. Emerging evidences suggest that the supercomplexes which further consist of multiple respiratory complexes play important role in regulating OXPHOS function. Dysfunction of the respiratory chain and its regulation has been implicated in various human diseases including neurodegenerative diseases and muscular disorders. Many mouse models have been established which exhibit mitochondrial defects in brain and muscles. Protocols presented here aim to help to analyze the structures of mitochondrial respiratory chain which include the preparation of the tissue samples, isolation of mitochondrial membrane proteins, and analysis of their respiratory complexes by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) in particular.

Keywords:  Assembly; Blue Native Gel; Brain; Muscle; Respiratory complex

DOI:  https://doi.org/10.1007/978-1-0716-2309-1_7
Bioessays. 2022 Jun 26. e2200027

Unraveling docking and initiation of mRNA export through the nuclear pore complex.

Mark Tingey, Weidong Yang.

The nuclear export of mRNA through the nuclear pore complex (NPC) is a process required for the healthy functioning of human cells, making it a critical area of research. However, the geometries of mRNA and the NPC are well below the diffraction limit of light microscopy, thereby presenting significant challenges in evaluating the discrete interactions and dynamics involved in mRNA nuclear export through the native NPC. Recent advances in biotechnology and single-molecule super-resolution light microscopy have enabled researchers to gain granular insight into the specific contributions made by discrete nucleoporins in the nuclear basket of the NPC to the export of mRNA. Specifically, by expanding upon the docking step facilitated by the protein TPR in the nuclear basket as well as identifying NUP153 as being the primary nuclear basket protein initiating export through the central channel of the NPC.

DOI: https://doi.org/10.1002/bies.202200027
Methods Mol Biol. 2022 ;2497 243-254

In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.

Arianna Montanari.

  I describe here a protocol for the analysis of mitochondrial protein synthesis as a useful tool to characterize the mitochondrial defects associated with mutations in mitochondrial tRNA genes. The yeast Saccharomyces cerevisiae mutants, bearing human equivalent pathogenic mutations, were used as a simple model for analysis. The mitochondrial proteins were labeled by L[35S]-methionine incorporation in growing cells, extracted from purified mitochondria, and fractionated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. By this method, it is possible to distinguish different protein synthesis profiles in the analyzed mitochondrial tRNA mutants.

Keywords:  Human equivalent mutations; In vivo L[35S]-methionine labeling; Mitochondria; Mitochondrial protein synthesis; Mitochondrial tRNA mutants; Saccharomyces cerevisiae

DOI:  https://doi.org/10.1007/978-1-0716-2309-1_15
Bioinformatics. 2022 Jun 27. pii: btac426. [Epub ahead of print]

Vfold-Pipeline: a web server for RNA 3D structure prediction from sequences.

Jun Li, Sicheng Zhang, Dong Zhang, Shi-Jie Chen.

SUMMARY: RNA 3D structures are critical for understanding their functions and for RNA-targeted drug design. However, experimental determination of RNA 3D structures is laborious and technically challenging, leading to the huge gap between the number of sequences and the availability of RNA structures. Therefore, the computer-aided structure prediction of RNA 3D structures from sequences becomes a highly desirable solution to this problem. Here, we present a pipeline server for RNA 3D structure prediction from sequences that integrates the Vfold2D, Vfold3D, and VfoldLA programs. The Vfold2D program can incorporate the SHAPE experimental data in 2D structure prediction. The pipeline can also automatically extract 2D structural constraints from the Rfam database. Furthermore, with a significantly expanded 3D template database for various motifs, this Vfold-Pipeline server can efficiently return accurate 3D structure predictions or reliable initial 3D structures for further refinement.
AVAILABILITY AND IMPLEMENTATION: http://rna.physics.missouri.edu/vfoldPipeline/index.html.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btac426
Methods Mol Biol. 2022 ;2507 19-39

Membrane Protein Production in Escherichia coli: Protocols and Rules.

Jordi Royes, Pauline Talbot, Christel Le Bon, Karine Moncoq, Marc Uzan, Francesca Zito, Bruno Miroux.

  Despite recent progresses in the use of eukaryotic expression system, production of membrane proteins for structural studies still relies on microbial expression systems. In this review, we provide protocols to achieve high level expression of membrane proteins in Escherichia coli, especially using the T7 RNA polymerase based expression system. From the design of the construct, the choice of the appropriate vector-host combination, the assessment of the bacterial fitness, to the selection of bacterial mutant adapted to the production of the target membrane protein, the chapter covers all necessary methods for a rapid optimization of a specific target membrane protein. In addition, we provide a protocol for membrane protein solubilization based on our recent analysis of the Protein Data Bank.

Keywords:  E. coli; Membrane proliferation; Membrane proteins; Production of recombinant proteins; T7 RNA polymerase

DOI:  https://doi.org/10.1007/978-1-0716-2368-8_2
Front Genet. 2022 ;13 898083

Signal Recognition Particle in Human Diseases.

Morgana K Kellogg, Elena B Tikhonova, Andrey L Karamyshev.

  The signal recognition particle (SRP) is a ribonucleoprotein complex with dual functions. It co-translationally targets proteins with a signal sequence to the endoplasmic reticulum (ER) and protects their mRNA from degradation. If SRP is depleted or cannot recognize the signal sequence, then the Regulation of Aberrant Protein Production (RAPP) is activated, which results in the loss of secretory protein mRNA. If SRP recognizes the substrates but is unable to target them to ER, they may mislocalize or degrade. All these events lead to dramatic consequence for protein biogenesis, activating protein quality control pathways, and creating pressure on cell physiology, and might lead to the pathogenesis of disease. Indeed, SRP dysfunction is involved in many different human diseases, including: congenital neutropenia; idiopathic inflammatory myopathy; viral, protozoal, and prion infections; and cancer. In this work, we analyze diseases caused by SRP failure and discuss their possible molecular mechanisms.

Keywords:  cancer; disease; protein quality control; protein sorting; protein targeting and transport; ribosome; signal recognition particle (SRP); translational control

DOI:  https://doi.org/10.3389/fgene.2022.898083
J Exp Biol. 2022 Jun 15. pii: jeb242612. [Epub ahead of print]225(12):

How does mitochondrial function relate to thermogenic capacity and basal metabolic rate in small birds?

Myriam S Milbergue, François Vézina, Véronique Desrosiers, Pierre U Blier.

  We investigated the role of mitochondrial function in the avian thermoregulatory response to a cold environment. Using black-capped chickadees (Poecile atricapillus) acclimated to cold (-10°C) and thermoneutral (27°C) temperatures, we expected to observe an upregulation of pectoralis muscle and liver respiratory capacity that would be visible in mitochondrial adjustments in cold-acclimated birds. We also predicted that these adjustments would correlate with thermogenic capacity (Msum) and basal metabolic rate (BMR). Using tissue high-resolution respirometry, mitochondrial performance was measured as respiration rate triggered by proton leak and the activity of complex I (OXPHOSCI) and complex I+II (OXPHOSCI+CII) in the liver and pectoralis muscle. The activity of citrate synthase (CS) and cytochrome c oxidase (CCO) was also used as a marker of mitochondrial density. We found 20% higher total CS activity in the whole pectoralis muscle and 39% higher total CCO activity in the whole liver of cold-acclimated chickadees relative to that of birds kept at thermoneutrality. This indicates that cold acclimation increased overall aerobic capacity of these tissues. Msum correlated positively with mitochondrial proton leak in the muscle of cold-acclimated birds while BMR correlated with OXPHOSCI in the liver with a pattern that differed between treatments. Consequently, this study revealed a divergence in mitochondrial metabolism between thermal acclimation states in birds. Some functions of the mitochondria covary with thermogenic capacity and basal maintenance costs in patterns that are dependent on temperature and body mass.

Keywords:   M sum ; Birds; Cold acclimation; LEAK; Metabolic intensity; Mitochondria; Oxidative phosphorylation

DOI:  https://doi.org/10.1242/jeb.242612
Nat Commun. 2022 Jun 29. 13(1): 3751

Pervasive translation of circular RNAs driven by short IRES-like elements.

Xiaojuan Fan, Yun Yang, Chuyun Chen, Zefeng Wang.

Some circular RNAs (circRNAs) were found to be translated through IRES-driven mechanism, however the scope and functions of circRNA translation are unclear because endogenous IRESs are rare. To determine the prevalence and mechanism of circRNA translation, we develop a cell-based system to screen random sequences and identify 97 overrepresented hexamers that drive cap-independent circRNA translation. These IRES-like short elements are significantly enriched in endogenous circRNAs and sufficient to drive circRNA translation. We further identify multiple trans-acting factors that bind these IRES-like elements to initiate translation. Using mass-spectrometry data, hundreds of circRNA-coded peptides are identified, most of which have low abundance due to rapid degradation. As judged by mass-spectrometry, 50% of translatable endogenous circRNAs undergo rolling circle translation, several of which are experimentally validated. Consistently, mutations of the IRES-like element in one circRNA reduce its translation. Collectively, our findings suggest a pervasive translation of circRNAs, providing profound implications in translation control.

DOI: https://doi.org/10.1038/s41467-022-31327-y