Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

Kerlavage, Anthony R.; Cooperman, Barry S.

doi:10.1021/bi00372a032

Cited by 23 publications

(19 citation statements)

References 44 publications

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“…The following methods were carried out as described previously (2-5): Millipore filter binding assay of noncovalent probe/subunit complex formation, localization of photoincorporation sites within 23 S rRNA by RNase H, and reverse transcriptase analyses. 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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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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“…70S ribosomes, 30S subunits, and 16S rRNA were prepared from E. coli Q13 cells as previously described (Kerlavage & Cooperman, 1986).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation and Purification of 30S Proteins. 30S proteins (TP30) were acetic acid extracted as previously described (Kerlavage & Cooperman, 1986). A combination of RP-HPLC and IE-HPLC was employed to prepare pure samples of proteins S3-S21.…”

Section: Methodsmentioning

confidence: 99%

Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

Buck¹,

Cooperman²

1990

Biochemistry

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In previous work we showed that on photolysis of Escherichia coli ribosomes in the presence of [3H]tetracycline (TC) the major protein labeled is S7, and we presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC [Goldman, R. A., Hasan, T., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1983) Biochemistry 22, 359-368]. In this work we use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNAPhe binding, the properties of the SPORE particles we obtain parallel very closely those measured earlier [Nomura, M., Mizushima, S., Ozaki, M., Traub, P., & Lowry, C. V. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 49-61], with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1396, in one of the major domains of the 30S subunit.

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“…We report here the sequences of amino acids in rat ribosomal proteins S27 and S29. Rat S29 is related to the prokaryotic family of S14 ribosomal proteins which latter can be cross-linked to puromycin (2) and, hence, are likely to be in the ribosomal Asite and involved in the binding of aminoacyl-tRNA. Rat…”

Section: Introductionmentioning

confidence: 99%

Zinc finger-like motifs in rat ribosomal proteins S27 and S29

et al. 1993

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The primary structures of the rat 40S ribosomal subunit proteins S27 and S29 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed by determination of amino acid sequences in the proteins. Ribosomal protein S27 has 83 amino acids and the molecular weight is 9,339. Hybridization of cDNA to digests of nuclear DNA suggests that there are 4-6 copies of the S27 gene; the mRNA for the protein is about 620 nucleotides in length. Ribosomal protein S29 has 55 amino acids and the molecular weight is 6,541. There are 14-17 copies of the S29 gene and its mRNA is about 500 nucleotides in length. Rat ribosomal protein S29 is related to several members of the archaebacterial and eubacterial S14 family of ribosomal proteins. S27 and S29 have zinc finger-like motifs as do other proteins from eukaryotic, archaebacterial, eubacterial, and mitochondrial ribosomes. Moreover, ribosomes and ribosomal subunits appear to contain zinc and iron as well.

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Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

Cited by 23 publications

References 44 publications

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

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

Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

Zinc finger-like motifs in rat ribosomal proteins S27 and S29

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