Structural and Functional Interactions of the tRNA-Ribosome Complex

Ofengand, James; Ciesiołka, Jerzy; Denman, Robert B.; Nurse, Kelvin

doi:10.1007/978-1-4612-4884-2_27

Cited by 25 publications

(21 citation statements)

References 89 publications

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“…In this report, we confirm that the polypeptide product of this open reading frame is a component of the mitochondrial large ribosomal subunit. This finding is particularly significant because there is abundant evidence from in vitro studies that E.coli L16 is located in the peptidyl transferase center (8,9) and is an important participant in the reconstitution of peptidyl transferase activity (10,11). Under certain conditions, however, reconstituted ribosomal particles lacking L16 are active in poly(Phe) synthesis (12) Purification of recombinant Rmll6p and production of polyclonal antisera A 167-amino-acid C-terminal fragment of Rmll6p was overexpressed from pCP211 in the E.coli BL21(DE3), pLysS.…”

Section: Introductionmentioning

confidence: 99%

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16

Pan¹,

Mason²

1995

Nucl Acids Res

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Section: Introductionmentioning

confidence: 99%

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16

Pan¹,

Mason²

1995

Nucl Acids Res

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“…Transfer RNAs (tRNAs) are the substrates used by ribosomes for synthesizing proteins+ To understand the mechanism of translation at the molecular level, it is essential to understand how tRNAs interact with the ribosome (Rheinberger et al+, 1981;Grajevskaja et al+, 1982;Kirillov et al+, 1983;Lill et al+, 1984)+ The anticodon arm of P-site tRNA interacts with mRNA in the vicinity of the cleft formed by the platform and the head of the 30S subunit (Lake, 1980;Gornicki et al+, 1984)+ The acceptor arms of A-site and P-site tRNAs interact with the peptidyl transferase center, which lies close to the base of the central protuberance of the 50S subunit (Ofengand, 1980;Olson et al+, 1982;Wower et al+, 1989)+ During peptide bond formation, the CCA-39 termini of A-site and P-site tRNAs must be proximal to allow peptidyl transfer, while their anticodons interact with adjacent codons on mRNA (Fairclough & Cantor, 1979a;Matzke et al+, 1980)+ Several models have been proposed for the arrangement of A-site and P-site tRNAs in the ribosome based on stereochemical considerations (Fuller & Hodgson, 1967;Woese, 1970;Rich, 1974;Sundaralingam et al+, 1975;Lake, 1977;Spirin & Lim, 1986;McDonald & Rein, 1987;Prabahakaran & Harvey, 1989;Nagano et al+, 1991;Easterwood et al+, 1994), fluorescence resonance energy transfer (FRET) (Johnson et al+, 1982;Paulsen et al+, 1983), crosslinking studies (Ofengand, 1980, Ofengand et al+, 1981Wower et al+, 1989Wower et al+, ,hotra et al+, 1998, and other methods (Spirin, 1983;Smith & Yarus, 1989;Wagenknecht et al+, 1989;Nierhaus et al+, 1998)+ Most of these models can be assigned to either the R (Rich, 1974) or S (Sundaralingam et al+, 1975) orientations+ In the R orientation, the T loop of A-site tRNA ...…”

Section: Introductionmentioning

confidence: 99%

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Joseph¹,

Whirl²,

Kondo³

et al. 2000

RNA

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The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 59 end of anticodon stem-loop analogs (ASLs) of tRNA and to the 59 end of deacylated tRNA Phe to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 458 between the respective planes of the molecules. The terminal phosphates of 39CCA are separated by 23 Å when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

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“…L27 has been identified as a constituent of the peptidyl transferase center on the basis of cross-linking experiments with either peptidyl-or aminoacyl-tRNA affinity labels and also with derivatives of antibiotics believed to act at the peptidyl transferase center (41). Immunoelectron microscopy has localized L27 on the central protuberance of the large subunit (29).…”

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confidence: 99%

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

Fearon¹,

Mason²

1988

Mol. Cell. Biol.

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The gene for MRP7, a 40-kilodalton protein of the large subunit of the yeast mitochondrial ribosome, was identified in a Agtll expression library by immunological screening with a monoclonal antibody to MRP7. An intact copy of MRP7 was then isolated from a yeast genomic library by colony hybridization. Gene disruption showed that MRP7 protein was essential for ribosomal function. Sequencing of MRP7 revealed a coding region for a basic (pI 10.6), 43.2-kilodalton protein containing 371 amino acid residues. Amino acid residues 28 to 112 of the deduced MRP7 sequence aligned with the 84 residues of the Escherichia coli ribosomal protein L27, but no significant similarity was detected between the carboxy-terminal 259 amino acids of MRP7 and other protein sequences in existing computer data bases. Within the aligned region, there was 49% amino acid identity between MRP7 and L27, compared with the 57% identity observed between L27 and its homolog in Bacilus stearothermophilus. The steady-state levels of the MRP7 protein and its mRNA were monitored in response to catabolite repression and to increased dosage of the MRP7 gene. The response to catabolite repression was characterized by a ninefold change in the level of the protein and little, if any, change in the level of the mRNA. In cells carrying the MRP7 gene on a high-copy-number plasmid, the mRNA was increased 20-fold, but there was no significant increase in MRP7 protein. Furthermore, MRP7 mRNA and protein accumulated at normal levels in [rho°] cells, which are devoid of 21S rRNA, indicating that the protein is relatively stable in the absence of ribosome assembly. Together, these results suggest that MRP7 is regulated posttranscriptionally, probably at the level of protein synthesis rather than protein turnover.Ribosomes are universal features of procaryotic and eucaryotic cells, and intensive investigation for over two decades has led to major advances in our understanding of their structure, function, synthesis, and evolution (for reviews, see references 16, 28, 31, 40, 44, and 66). Much of this knowledge has been developed in studies of the Escherichia coli ribosome, which has become the standard of comparison for the analysis of ribosomes in other organisms. In contrast to bacteria, eucaryotic cells maintain multiple, distinctly different ribosomal populations for protein synthesis in the cytoplasm, chloroplasts, and mitochondria. The presence of cytoplasmic and organellar ribosomes provides an opportunity to compare the properties of genetically separate sets of ribosomal components within a single cell and to establish relationships between these components and those of the E. coli ribosome. These comparisons should help to identify features of eucaryotic ribosomes that are special adaptations to the complex genomic organization and intracellular compartmentalization of eucaryotic cells. The ribosomes of mitochondria and chloroplasts are of special interest in this regard because the genes for the constituents of organellar ribosomes are divided between two...

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Structural and Functional Interactions of the tRNA-Ribosome Complex

Cited by 25 publications

References 89 publications

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

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