A Photolabile Oligodeoxyribonucleotide Probe of the Decoding Site in the Small Subunit of the Escherichia coli Ribosome: Identification of Neighboring Ribosomal Components

Muralikrishna, Parimi; Cooperman, Barry S.

doi:10.1021/bi00172a015

Cited by 19 publications

(14 citation statements)

References 42 publications

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“…Ribosomal protein-mapping studies indicate that E. coli S21 protein is in the platform region of the small subunit, the site of Shine-Dalgarno and codon-anticodon interactions (8). S21 is in very close proximity to both the 16S rRNA and the initiation region of mRNAs, as shown by cross-linking and resonance energy transfer experiments (15,21,48). However, the in vivo function of S21 has not been studied genetically.…”

Section: Discussionmentioning

confidence: 99%

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Green-Willms

Fox

Costanzo

1998

Molecular and Cellular Biology

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Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5 untranslated leaders (5-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5-UTLs which, in conjunction with mRNAspecific translational activation, is required for organellar translation initiation.While mitochondrial ribosomes exhibit distinct similarities to bacterial (eubacterial) ribosomes (30, 69), the yeast mitochondrial translation system has many intriguing differences from bacterial systems. Mitochondrial ribosomes have more proteins than do bacterial ribosomes (23, 43). Of the mitochondrial ribosomal proteins whose sequences are known, some are simple homologs of their bacterial counterparts, others have domains homologous to bacterial ribosomal proteins attached to domains with no recognizable homology to any known proteins, and still others are completely unrelated to bacterial ribosomal proteins (reviewed in reference 32). Saccharomyces cerevisiae mitochondrial mRNAs generally have long, AϩU-rich 5Ј untranslated leaders (5Ј-UTLs) lacking a typical Shine-Dalgarno sequence (11,27,31). While the mechanism of start site selection remains obscure in this system, translation initiation on most or all yeast mitochondrial mRNAs requires membrane-bound mRNA-specific activator proteins whose targets lie in the 5Ј-UTLs (reviewed in reference 27). These mRNA-specific activators appear to play a dual role in mitochondrial gene expression: tethering the synthesis of the very hydrophobic mitochondrial gene products to the inner membrane (27) and modulating the translation levels of individual mRNAs (63).We have focused on translation of the COX2 and COX3 mRNAs, which encode subunits II and III of cytochrome c oxidase, respectively. Previous studies have identified their mRNA-specific translation...

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Section: Discussionmentioning

confidence: 99%

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Green-Willms

Fox

Costanzo

1998

Molecular and Cellular Biology

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“…Proteins were isolated from labeled 50 S subunits by acetic acid extraction and acetone precipitation in the usual fashion (14). Labeled proteins were identified by RP-HPLC (15), SDS-PAGE coupled with autoradiography (3)(4)(5), and, as needed, agarose antibody affinity chromatography (16).…”

Section: Methodsmentioning

confidence: 99%

Ribosomal Proteins Neighboring 23 S rRNA Nucleotides 803−811 within the 50 S Subunit

Alexander

Cooperman²

1998

Biochemistry

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We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50 S ribosomal subunit components neighboring its target site, nucleotides 803-811 in 23 S rRNA. Photolysis of the complex formed between the probe and 50 S subunits leads to site-specific probe photoincorporation into proteins L15, L17, and L20, labeled to greater extents, and L13 and L21, labeled to lesser extents. Portions of each of these proteins thus lie within 23 A of nucleotide U803. These results lead us to conclude that nucleotides 803-811 fall on the side of the L13-L17-L20-L21 protein cluster [Walleczek et al. (1988) EMBO J. 7, 3571-3576] that points from the back of the 50 S particle toward the peptidyl transferase center within the 50 S subunit. Such placement is consistent with the observation that an oligoDNA probe directed to nucleotides 803-811 decreases P-site binding of tRNA [Hill et al. (1990) Biochim. Biophys. Acta 1050, 45-50].

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“…We have been using radioactive photolabile oligonucleotide probes (PHONTs) targeted toward functionally important rRNA sites to identify proteins and individual nucleotides sitespecifically photolabeled from these sites as a way of generating crosslinking information useful for the development of detailed structural models (15)(16)(17)(18)(19)(20)(21). The PHONT approach offers several important advantages for such an exercise.…”

Section: Introductionmentioning

confidence: 99%

Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit

Bukhtiyarov

Druzina

Cooperman

1999

Nucleic Acids Research

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We report the synthesis of a radioactive, photolabile 2'-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called 'P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 A from 23S RNA nucleotides G2252 and G2253. PHONT1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.

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A Photolabile Oligodeoxyribonucleotide Probe of the Decoding Site in the Small Subunit of the Escherichia coli Ribosome: Identification of Neighboring Ribosomal Components

Cited by 19 publications

References 42 publications

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Ribosomal Proteins Neighboring 23 S rRNA Nucleotides 803−811 within the 50 S Subunit

Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit

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