Ribosome structural dynamics in translocation: yet another functional role for ribosomal RNA

2017 ◽  
Vol 50 ◽  
Author(s):  
Harry F. Noller ◽  
Laura Lancaster ◽  
Srividya Mohan ◽  
Jie Zhou
Keyword(s):  
Protein Synthesis ◽  
Structural Dynamics ◽  
Ribosomal Rna ◽  
Messenger Rna ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Detailed Structural Analysis ◽  
Ribonucleoprotein Complexes ◽  
Elongation Phase ◽  
Polypeptide Chains

AbstractRibosomes are remarkable ribonucleoprotein complexes that are responsible for protein synthesis in all forms of life. They polymerize polypeptide chains programmed by nucleotide sequences in messenger RNA in a mechanism mediated by transfer RNA. One of the most challenging problems in the ribosome field is to understand the mechanism of coupled translocation of mRNA and tRNA during the elongation phase of protein synthesis. In recent years, the results of structural, biophysical and biochemical studies have provided extensive evidence that translocation is based on the structural dynamics of the ribosome itself. Detailed structural analysis has shown that ribosome dynamics, like aminoacyl-tRNA selection and catalysis of peptide bond formation, is made possible by the properties of ribosomal RNA.

scholarly journals The parable of the caveman and the Ferrari: protein synthesis and the RNA world

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160187 ◽  
Author(s):  
Harry F. Noller
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Rna ◽  
Rna World ◽  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Ribonucleoprotein Particle ◽  
Supportive Role

The basic steps of protein synthesis are carried out by the ribosome, a very large and complex ribonucleoprotein particle. In keeping with its proposed emergence from an RNA world, all three of its core mechanisms—aminoacyl-tRNA selection, catalysis of peptide bond formation and coupled translocation of mRNA and tRNA—are embodied in the properties of ribosomal RNA, while its proteins play a supportive role. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals Crystal structure of eukaryotic ribosome and its complexes with inhibitors

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160184 ◽  
Author(s):  
Gulnara Yusupova ◽  
Marat Yusupov
Keyword(s):  
Crystal Structure ◽  
Ribosomal Rna ◽  
Protein Biosynthesis ◽  
Protein Translation ◽  
Peptide Bond ◽  
Binding Mode ◽  
High Eukaryote ◽  
Peptide Bond Formation ◽  
80S Ribosome ◽  
Eukaryotic Ribosome

A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome—the high-eukaryote–specific long ribosomal RNA segments (about 1MDa)—remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

2009 ◽  
Vol 6 (suppl_5) ◽  
Author(s):  
Ada Yonath
Keyword(s):  
Synchrotron Radiation ◽  
Genetic Code ◽  
Structural Information ◽  
Peptide Bond ◽  
Polymerase Activity ◽  
Resistance Mechanisms ◽  
Peptide Bond Formation ◽  
Peptide Bonds ◽  
Polypeptide Chains

Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric pre-biotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

scholarly journals Structural basis for the context-specific action of classic peptidyl transferase inhibitors.

2021 ◽  
Author(s):  
Egor A. Syroegin ◽  
Laurin Flemmich ◽  
Dorota S Klepacki ◽  
Nora S Vazquez-Laslop ◽  
Ronald Micura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Peptide Bond ◽  
Amino Acid Sequences ◽  
Structural Basis ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Inhibit Protein Synthesis ◽  
Clinically Significant ◽  
Context Specific

Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.

scholarly journals Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Benjamin D Hobson ◽  
Linghao Kong ◽  
Erik W Hartwick ◽  
Ruben L Gonzalez ◽  
Peter A Sims
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Acyl Transfer ◽  
Proximity Ligation Assay ◽  
Nascent Polypeptide ◽  
Rna Translation ◽  
Biochemical Assays ◽  
Messenger Rna Translation ◽  
Polypeptide Chains

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

scholarly journals A Single Polyphosphate Kinase-Based NTP Regeneration System Driving Cell-Free Protein Synthesis

2019 ◽  
Author(s):  
Po-Hsiang Wang ◽  
Kosuke Fujishima ◽  
Samuel Berhanu ◽  
Yutetsu Kuruma ◽  
Tony Z. Jia ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Bond Formation ◽  
Peptide Bond ◽  
Functional Expression ◽  
Protein Yield ◽  
Polyphosphate Kinase ◽  
Peptide Bond Formation ◽  
Regeneration System ◽  
Free Protein ◽  
Cell Free Protein Synthesis

<p>Current reconstituted cell-free protein synthesis systems regenerate the ATP and GTP required for peptide bond formation using phosphocreatine and three coupled kinase reactions. Here, we replace the three-kinase system with a single polyphosphate kinase, resulting in a comparable protein yield and enabling functional expression of a heat-sensitive enzyme at 37°C.</p>

scholarly journals Origins of the Mechanochemical Coupling of Peptide Bond Formation to Protein Synthesis

2018 ◽  
Vol 140 (15) ◽  
pp. 5077-5087 ◽  
Author(s):  
Benjamin Fritch ◽  
Andrey Kosolapov ◽  
Phillip Hudson ◽  
Daniel A. Nissley ◽  
H. Lee Woodcock ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Mechanochemical Coupling
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