Optimization of Ribosome Structure and Function by rRNA Base Modification (original) (raw)
Related papers
RNA biology, 2016
rRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2'-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in dise...
Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly
Nature structural & molecular biology, 2015
We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.
Expanding the Nucleotide Repertoire of the Ribosome with Post-Transcriptional Modifications
ACS Chemical Biology, 2007
In all kingdoms of life, RNAs undergo specific post-transcriptional modifications. More than 100 different analogues of the four standard RNA nucleosides have been identified. Modifications in ribosomal RNAs are highly prevalent and cluster in regions of the ribosome that have functional importance, a high level of nucleotide conservation, and that typically lack proteins. Modifications also play roles in determining antibiotic resistance or sensitivity. A wide spectrum of chemical diversity from the modifications provides the ribosome with a broader range of possible interactions between ribosomal RNA regions, transfer RNA, messenger RNA, proteins, or ligands by influencing local ribosomal RNA folds and fine-tuning the translation process. The collective importance of the modified nucleosides in ribosome function has been demonstrated for a number of organisms, and further studies may reveal how the individual players regulate these functions through synergistic or cooperative effects. In all kingdoms of life, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other RNAs undergo specific post-transcriptional modification by a wide variety of enzymes (1). To date, >100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (2). These modifications can be organized into four main types (Figure 1) (1). The first involves isomerization of uridine to pseudouridine (5-ribosyluracil, Ψ), which contains a C-rather than the typical N-glycosidic linkage, as well as an additional imino group that is available for unique hydrogen-bonding interactions. The second includes alterations to the bases, such as methylation (typically on carbon, primary nitrogen, or tertiary nitrogen), deamination (e.g., inosine), reduction (e.g., dihydrouridine), thiolation, or alkylation (e.g., isopentenylation or threonylation). The third involves methylation of the ribose 2′ hydroxyl (Nm). The fourth type includes more complex modifications, such as multiple modifications (e.g., 5-methylaminomethyl-2-thiouridine; 3-(3-amino-3-carboxypropyl)uridine, acp 3 U; 1-methyl-3-(3-amino-3carboxypropyl)pseudouridine, m 1 acp 3 Ψ) or "hypermodifications" that can be incorporated by specific exchange mechanisms (e.g., queuosine). The possible electronic and steric effects of the nucleoside modifications on base pairing, base stacking, and sugar pucker in RNA have been discussed in detail by Davis (3) and Agris (4), among others (1). The effects of modifications such as Ψ on RNA hydration and dynamics have also been considered (4). Modified nucleotides in the ribosome are varied in their identity, but highly localized in their positions (5). If the sites of modification are mapped on the secondary structures of the small and large subunit (SSU and LSU) rRNAs, they might appear to be random; however, if the same modifications are located within the ribosome tertiary structures from high-resolution Xray crystal structures (6,7), they occur in the most functionally important regions (Figure 2). In particular, regions dedicated to peptidyl transfer, such as the A and P sites, the polypeptide
Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria
Nucleic Acids Research, 2007
Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function.
Delayed rRNA Processing Results in Significant Ribosome Biogenesis and Functional Defects
Molecular and Cellular Biology, 2003
mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed ؊1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at ؊1 frameshift signals, promoting increased programmed ؊1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus. The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis.
Non-Coding RNA, 2021
Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of t...
Functional Defects Significant Ribosome Biogenesis and Delayed rRNA Processing Results in
mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed ؊1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at ؊1 frameshift signals, promoting increased programmed ؊1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus. The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis.
The Ribosome: A biochemist's mechano set
Biochemistry and Cell Biology, 1985
A ribosome, the cellular site for protein synthesis, is a very complex organelle composed of a myriad of macromolecular substructures. As models for this complex structure, we have been examining the structures and interactions of eukaryotic 5S and 5.8S rRNAs using adaptations of rapid RNA gel sequencing techniques. Estimates for their higher order structures have been proposed or evaluated, sites of interaction with other ribosomal components have been delineated, and the topography of these RNAs within the intact ribosome or 60S subunit have been examined. The results indicate that a universal structure for the ribosomal RNAs may only be present within the ribosome, that these molecules are probably present, at least in part, within the ribosomal interface, and that the bases for interactions with other ribosomal components are strongly dependent on their higher order structure. The experimental approaches which underlie these studies are considered in this review and the signific...