Structure of the Ribosome at 5.5 A Resolution and Its Interactions with Functional Ligands

Noller, Harry F.; Yusupov, Marat; Yusupova, G.; Baucom, Albion; Lieberman, Kate R.; Lancaster, Laura; Dallas, Anne; Fredrick, Kurt; Earnest, Thomas; Cate, J.H.D.

doi:10.1101/sqb.2001.66.57

Cited by 22 publications

(16 citation statements)

References 48 publications

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“…It could be that each fragment binds the A site in a slightly different way, because the rest of the aa-tRNA is not present to aid in proper positioning. Although the binding of both deacylated tRNAs and CpAaa derivatives to the A site is consistent with the chemical footprinting data acquired using intact aa-tRNAs (Nissen et al 2000;Noller et al 2001), these fragments could bind in position that was only 1 or 2 Å different from the whole aatRNA. Such small differences in positioning could be sufficient to lead to different binding affinities for the fragments and would lead to the erroneous conclusion that specificity was present.…”

supporting

confidence: 80%

Binding of misacylated tRNAs to the ribosomal A site

Dale

Uhlenbeck

2005

RNA

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To test whether the ribosome displays specificity for the esterified amino acid and the tRNA body of an aminoacyl-tRNA (aatRNA), the stabilities of 4 correctly acylated and 12 misacylated tRNAs in the ribosomal A site were determined. By introducing the GAC (valine) anticodon into each tRNA, a constant anticodonÁcodon interaction was maintained, thus removing concern that different anticodonÁcodon strengths might affect the binding of the different aa-tRNAs to the A site. Surprisingly, all 16 aatRNAs displayed similar dissociation rate constants from the A site. These results suggest that either the ribosome is not specific for different amino acids and tRNA bodies when intact aa-tRNAs are used or the specificity for the amino acid side chain and tRNA body is masked by a conformational change upon aa-tRNA release.Keywords: tRNA; ribosome; uniformity; misacylated tRNA; A site; ribosome binding Several studies indicate that the rate of incorporation of an amino acid into a growing polypeptide chain in Escherichia coli is roughly uniform when the substrate, aa-tRNAÁEF-TuÁGTP ternary complex, is saturating. For example, Curran and Yarus (1989) evaluated 18 elongator aminoacyl-tRNAs (aa-tRNAs) in their ability to compete with a frameshift and concluded that the rates of incorporation of aa-tRNAs belonging to codons used frequently in highly expressed genes were all near the average aa-tRNA selection rate, while aa-tRNAs that were not preferred in highly expressed genes were incorporated faster or slower than the mean. This observation supports the proposal made by Grosjean and Fiers (1982), who suggested that codons used in highly expressed genes are selected for maintenance of a uniform anticodonÁcodon interaction strength. Furthermore, Pedersen (1984) found that, in three different growth media, the translation rates of several different mRNAs varied by less than twofold, and that the small differences observed were due to the low concentration of aa-tRNAs belonging to rare codons. Thus, while the lower concentration of rare aatRNAs (and thus ternary complexes) can lead to slower translation rates, it appears that all aa-tRNAs are used in an equivalent manner during the translation of highly expressed genes.Not only does the overall rate of translation appear to be uniform, but there is accumulating biochemical evidence that several of the individual steps of translation elongation also display uniformity. It has been shown that the GTPbound form of elongation factor Tu (EF-TuÁGTP) binds all elongator aa-tRNAs with similar affinities (Louie et al. 1984;Ott et al. 1990). In addition, recent studies by have demonstrated that eight different purified aa-tRNAs bind the ribosomal A and P sites with a very small range of K D values. Finally, kinetic studies have shown that Phe-tRNA Phe and Trp-tRNA Trp have similar rates of GTP hydrolysis and peptide bond formation (Pape et al. 1998;Cochella and Green 2005).The manner in which uniformity is achieved is difficult to understand given the substantial diversity amon...

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supporting

confidence: 80%

Binding of misacylated tRNAs to the ribosomal A site

Dale

Uhlenbeck

2005

RNA

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“…The ribosome is a megadalton complex, composed of multiple proteins and RNAs, in which the activities of numerous functional centers are coordinated to synthesize proteins with great accuracy. In simple terms, the ribosome orchestrates the various stages in peptide synthesis by coordinating the activities of least nine functional centers through at least seven discrete unidirectional steps (reviewed in Noller et al 2001). Analyses of static X-ray crystal structures have been useful in localizing the functional centers at the atomic level revealing, for example, that the catalytic activity of the ribosome is mediated by RNA, and identifying the binding sites for antibiotics (reviewed in Yonath et al 1998;Noller et al 2001;Steitz and Moore 2003;Wilson and Nierhaus 2003).…”

Section: Introductionmentioning

confidence: 99%

“…In simple terms, the ribosome orchestrates the various stages in peptide synthesis by coordinating the activities of least nine functional centers through at least seven discrete unidirectional steps (reviewed in Noller et al 2001). Analyses of static X-ray crystal structures have been useful in localizing the functional centers at the atomic level revealing, for example, that the catalytic activity of the ribosome is mediated by RNA, and identifying the binding sites for antibiotics (reviewed in Yonath et al 1998;Noller et al 2001;Steitz and Moore 2003;Wilson and Nierhaus 2003). Cryo-EM studies have supplied complementary information, providing dynamic views of intra-ribosomal movements during many of the different phases of the translation cycle (reviewed in Frank 2003).…”

Section: Introductionmentioning

confidence: 99%

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

et al. 2005

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Abstract5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.

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“…The regions of 16S rRNA, which play a role in forming these intersubunit bridges are known in both E. coli and T. thermophilus from biochemical and crystallographic methods with many of the bridge regions shared (Chapman and Noller 1977;Noller et al 2001;Schuwirth et al 2005), therefore it is assumed for this work that subunit bridge regions are similar in all organism tested. Four helices known to be important for subunit bridge formation, helices 20, 23, 24, and 27 (helix numbers from Sykes and Williamson [2009]), show RNase T1 cleavage patterns in at least one of the organisms tested.…”

Section: Functional Regions Of 16s Rrna Are Not Appropriately Structumentioning

confidence: 99%

Analysis of r-protein and RNA conformation of 30S subunit intermediates in bacteria

Napper

Culver

2015

RNA

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The ribosome is a large macromolecular complex that must be assembled efficiently and accurately for the viability of all organisms. In bacteria, this process must be robust and tunable to support life in diverse conditions from the ice of arctic glaciers to thermal hot springs. Assembly of the Small ribosomal SUbunit (SSU) of Escherichia coli has been extensively studied and is highly temperature-dependent. However, a lack of data on SSU assembly for other bacteria is problematic given the importance of the ribosome in bacterial physiology. To broaden the understanding of how optimal growth temperature may affect SSU assembly, in vitro SSU assembly of two thermophilic bacteria, Geobacillus kaustophilus and Thermus thermophilus, was compared with that of E. coli. Using these phylogenetically, morphologically, and environmentally diverse bacteria, we show that SSU assembly is highly temperature-dependent and efficient SSU assembly occurs at different temperatures for each organism. Surprisingly, the assembly landscape is characterized by at least two distinct intermediate populations in the organisms tested. This novel, second intermediate, is formed in the presence of the full complement of r-proteins, unlike the previously observed RI * particle formed in the absence of late-binding r-proteins in E. coli. This work reveals multiple distinct intermediate populations are present during SSU assembly in vitro for several bacteria, yielding insights into RNP formation and possible antimicrobial development toward this common SSU target.

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Structure of the Ribosome at 5.5 A Resolution and Its Interactions with Functional Ligands

Cited by 22 publications

References 48 publications

Binding of misacylated tRNAs to the ribosomal A site

Binding of misacylated tRNAs to the ribosomal A site

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

Analysis of r-protein and RNA conformation of 30S subunit intermediates in bacteria

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