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Journal of Applied Physiology... Aug 2019Skeletal muscle mass responds in a remarkable manner to alterations in loading and use. It has long been clear that skeletal muscle hypertrophy can be prevented by... (Review)
Review
Skeletal muscle mass responds in a remarkable manner to alterations in loading and use. It has long been clear that skeletal muscle hypertrophy can be prevented by inhibiting RNA synthesis. Since 80% of the cell's total RNA has been estimated to be rRNA, this finding indicates that de novo production of rRNA via transcription of the corresponding genes is important for such hypertrophy to occur. Transcription of rDNA by RNA Pol I is the rate-limiting step in ribosome biogenesis, indicating in turn that this biogenesis strongly influences the hypertrophic response. The present minireview focuses on ) a brief description of the key steps in ribosome biogenesis and the relationship of this process to skeletal muscle mass and ) the coordination of ribosome biogenesis and protein synthesis for growth or atrophy, as exemplified by the intracellular AMPK and mTOR pathways.
Topics: Animals; Humans; Hypertrophy; Muscle, Skeletal; Muscular Diseases; Protein Biosynthesis; RNA, Ribosomal; Ribosomes; Transcription, Genetic
PubMed: 31219775
DOI: 10.1152/japplphysiol.00963.2018 -
Physical Review. E Jan 2022The impact of ribosome exit tunnel electrostatics on the protein elongation rate or on forces acting upon the nascent polypeptide chain are currently not fully...
The impact of ribosome exit tunnel electrostatics on the protein elongation rate or on forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in rabbit reticulocytes. Here we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show that a strong electrostatic screening is due to water molecules (not mobile ions) attracted to the ribosomal nucleic acid phosphate moieties buried in the immediate vicinity of the tunnel wall. We also show how the tunnel wall components and local ribosomal protein protrusions impact on the electrostatic potential profile and impede charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of -40% to +85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derive, at single-residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall, shape, and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, x-ray crystallography, physical chemistry, biomechanics, and synthetic and multiomics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control, and the role of the ribosome's mechanochemistry in the cotranslational protein folding.
Topics: Animals; Peptides; Protein Biosynthesis; Protein Folding; Rabbits; Ribosomes; Static Electricity
PubMed: 35193250
DOI: 10.1103/PhysRevE.105.014409 -
PloS One 2020In the past two decades, research into the biochemical, biophysical and structural properties of the ribosome have revealed many different steps of protein translation....
In the past two decades, research into the biochemical, biophysical and structural properties of the ribosome have revealed many different steps of protein translation. Nevertheless, a complete understanding of how they lead to a rapid and accurate protein synthesis still remains a challenge. Here we consider a coarse network analysis in the bacterial ribosome formed by the connectivity between ribosomal (r) proteins and RNAs at different stages in the elongation cycle. The ribosomal networks are found to be dis-assortative and small world, implying that the structure allows for an efficient exchange of information between distant locations. An analysis of centrality shows that the second and fifth domains of 23S rRNA are the most important elements in all of the networks. Ribosomal protein hubs connect to much fewer nodes but are shown to provide important connectivity within the network (high closeness centrality). A modularity analysis reveals some of the different functional communities, indicating some known and some new possible communication pathways Our mathematical results confirm important communication pathways that have been discussed in previous research, thus verifying the use of this technique for representing the ribosome, and also reveal new insights into the collective function of ribosomal elements.
Topics: Bacteria; Computational Biology; Gene Regulatory Networks; Protein Biosynthesis; RNA, Ribosomal, 23S; Ribosomal Proteins; Ribosomes; Transcription Elongation, Genetic
PubMed: 33017414
DOI: 10.1371/journal.pone.0239700 -
Nucleic Acids Research Feb 2020Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel... (Review)
Review
Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and extract-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed.
Topics: Amino Acids; Bioengineering; Protein Biosynthesis; RNA, Transfer; Ribosomal Proteins; Ribosomes
PubMed: 31777928
DOI: 10.1093/nar/gkz1011 -
Nature Communications May 2023In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal...
In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.
Topics: Cryoelectron Microscopy; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes
PubMed: 37169754
DOI: 10.1038/s41467-023-38161-w -
Nucleic Acids Research Sep 2022Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of...
Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the 'waste' rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF.
Topics: Humans; RNA, Messenger; RNA, Ribosomal; Ribosomal Proteins; Ribosomes
PubMed: 35687114
DOI: 10.1093/nar/gkac484 -
Current Opinion in Microbiology Feb 2024Sensing small molecules is crucial for microorganisms to adapt their genetic programs to changes in their environment. Arrest peptides encoded by short regulatory... (Review)
Review
Sensing small molecules is crucial for microorganisms to adapt their genetic programs to changes in their environment. Arrest peptides encoded by short regulatory open reading frames program the ribosomes that translate them to undergo translational arrest in response to specific metabolites. Ribosome stalling in turn controls the expression of downstream genes on the same messenger RNA by translational or transcriptional means. In this review, we present our current understanding of the mechanisms by which ribosomes translating arrest peptides sense different metabolites, such as antibiotics or amino acids, to control gene expression.
Topics: Protein Biosynthesis; Ribosomes; Peptides; Anti-Bacterial Agents
PubMed: 38159358
DOI: 10.1016/j.mib.2023.102418 -
The Journal of Antimicrobial... Apr 2020This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we... (Review)
Review
This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we have examined the effect of 52 different antibiotics on ribosomal subunit formation in six different microorganisms. Most of the antimicrobials we have studied are specific, preventing the formation of only the subunit to which they bind. A few interesting exceptions have also been observed. Forty-one research publications and a book chapter have resulted from this investigation. This review will describe the methodology we used and the fit of our results to a hypothetical model. The model predicts that inhibition of subunit assembly and translation are equivalent targets for most of the antibiotics we have investigated.
Topics: Anti-Bacterial Agents; Bacteria; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 31942624
DOI: 10.1093/jac/dkz544 -
The Journal of Biological Chemistry Jun 2022Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the...
Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNA or Val-tRNA reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.
Topics: Peptide Biosynthesis; Peptides; Polylysine; RNA, Messenger; RNA, Transfer; RNA, Transfer, Lys; Ribosomes
PubMed: 35595100
DOI: 10.1016/j.jbc.2022.102039 -
Nucleic Acids Research May 2021Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits...
Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli. A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior during amino acid misincorporation, -1 or +1 frameshifting, stop codon readthrough, and internal translation initiation. Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.
Topics: Codon, Terminator; Frameshifting, Ribosomal; Peptide Chain Initiation, Translational; Peptide Chain Termination, Translational; Protein Biosynthesis; RNA, Transfer; Ribosomes
PubMed: 33950196
DOI: 10.1093/nar/gkab259