Photoinduced affinity labeling of the Escherichia coli ribosome puromycin site

Jaynes, E.N.; Grant, Paul G.; Giangrande, Giuseppe; Wieder, Robert; Cooperman, Barry S.

doi:10.1021/bi00597a001

Cited by 48 publications

(103 citation statements)

References 52 publications

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“…70 S ribosomes were prepared from both E. coli MRE600 [5] and E. coli Q13 [6] as described. Large scale preparations of 30 S and 50 S subunits were obtained by zonal centrifugation in a Beckman Ti 15 rotor essentially as in [7].…”

Section: Methodsmentioning

confidence: 99%

“…ethanol, redissolved in buffer and ethanolprecipitated again, and separated into subunits by centrifugation through a 5-20% sucrose gradient in a high salt buffer (50 mM Tris-HCl, 10 mM MgCl,, 50 mM KCl, 400 mM NaCl). The subunits were collected as in [6]. Protein was obtained by Mg'+-acetic acid extraction followed by acetone precipitation [20].…”

Section: Methodsmentioning

confidence: 99%

“…Areas of protein staining were circled on the sheet, and areas not staining were divided into a grid, as shown. Portions of the gel corresponding to the delineated areas were excised and counted for radioactivity as in [6]. The drawing is from a Xerox copy of the plastic sheet.…”

Section: Methodsmentioning

confidence: 99%

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Photoincorporation of tetracycline into Escherichia coli ribosomes

et al. 1980

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Photoincorporation of tetracycline into Escherichia coli ribosomes

et al. 1980

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“…Photoaffinity labeling of the ribosome has proved to be especially instructive in attempts to relate structure and function in this complex assemblage of proteins and nucleic acids (5)(6)(7)(8)(9). The photoinduced incorporation of puromycin into each of the ribosomal subunits in particular has been shown to be biochemically significant and highly specific (10)(11)(12)(13).…”

mentioning

confidence: 99%

“…MATERIALS AND METHODS Ribosomes and ribosomal subunits were isolated from E. coli TPR201, a kasugamycin-resistant strain lacking N6,N6-dimethyladenosine (24), by using methods previously described (11). The procedures for photolytic incorporation of [8-3H1-puromycin (Amersham) and the analysis of photolabeled products have also been described (10)(11)(12)(13) Antibodies were induced in rabbits by toe-pad injection of a conjugate of bovine serum albumin and N6;N6-dimethyladenosine (22,25).…”

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Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.

Olson¹,

Grant²,

Glitz³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Ribosomes from Escherichia coli strain TPR201 (which lack N6,N6-dimethyladenosine) have been photoaffinity labeled with [3Hlpuromycin in the presence of chloramphenicol. Puromycin-modified 30S ribosomal subunits appear to be identical to untreated subunits in electron micrographs and are efficiently precipitated by antibodies to the puromycin analog N6,N6-dimethyladenosine. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of 30S subunits which appear to be crosslinked by a single IgG molecule. A predominant site of puromycin photoaffinity labeling has been identified from the apparent point of contact of antibody and ribosomal subunit. The puromycin site is localized to the small upper portion of the particle on the side opposite to the subunit platform. This location is close to that reported for ribosomal protein S14, the major puromycin-labeled protein in the small ribosomal subunit.Photoaffinity labeling was first used by Westheimer and his colleagues (1) to identify amino acid residues involved in the active center of chymotrypsin. The technique has since become a major tool in the identification of biologically meaningful sites of interaction between ligands and macromolecules or complexes of macromolecules including enzymes, antibodies, transport proteins, receptors or other membrane proteins, nucleic acids, and nucleoproteins (reviewed in refs. 2-4). Photoaffinity labeling of the ribosome has proved to be especially instructive in attempts to relate structure and function in this complex assemblage of proteins and nucleic acids (5-9). The photoinduced incorporation of puromycin into each of the ribosomal subunits in particular has been shown to be biochemically significant and highly specific (10-13).Immunoelectron microscopy-the visualization in electron micrographs of antibody-linked biological structures-has also been of great value in defining ribosome structure and function. The technique has been used to localize individual proteins of the ribosomal subunits (14-20), to help identify a ribosomal neighborhood involved in the initiation of protein synthesis (15,16), to indicate the possible location of codon-anticodon interaction (21), and to localize a specific minor nucleotide (22) and the derivatized 3' end (23) of 16S ribosomal RNA.In the course of immunoelectron microscopic localization of N6,N6-dimethyladenosine (22), antibodies induced by a bovine serum albumin-dimethyladenosine complex were found to be highly specific. These antibodies showed almost no binding of adenosine or 5'-AMP. However, the antibodies were almost as reactive toward puromycin, a dimethyladenosine analog, as toward N6,N6-dimethyladenosine itself (22). In the study described here, we exploited this finding by combining the techniques of photoaffinity labeling and immunoelectron microscopy to identify the predominant site of puromycin binding on the Escherichia coli 30S ribosomal subunit. Our work provides direct visual evidence fo...

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The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Beauclerk

Cundliffe

1988

The EMBO Journal

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Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of ‘domain IV’ of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757‐1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2‐protected oligonucleotides, also derived from domain IV (i.e. from residues 1955‐2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.

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Photoinduced affinity labeling of the Escherichia coli ribosome puromycin site

Cited by 48 publications

References 52 publications

Photoincorporation of tetracycline into Escherichia coli ribosomes

Photoincorporation of tetracycline into Escherichia coli ribosomes

Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

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