The mechanism of chloramphenicol-induced changes in the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin. Evidence for puromycin and chloramphenicol sites on the 30S subunit

Gp, Grant; Bs, Cooperman; Wa, Strycharz

doi:10.1021/bi00578a004

Cited by 19 publications

(3 citation statements)

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“…Our results are important in showing the consistency of the analytical data that identify S14 as the major protein labeled by puromycin in the presence of chloramphenicol (12,13) and of the immunoelectron microscopic localization of protein S14 (14,19). But we also believe that the observations may have much greater significance.…”

Section: Discussionsupporting

confidence: 65%

“…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).…”

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“…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). A globulin fraction was isolated by ammonium sulfate precipitation (0-40% saturation fraction) and gel filtration on Ultrogel ACA22 (LKB).…”

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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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Section: Discussionsupporting

confidence: 65%

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confidence: 99%

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confidence: 99%

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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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Sequential hydration of dry proteins: A direct difference IR investigation of sequence homologs lysozyme and α‐lactalbumin

Poole

Finney

1984

Biopolymers

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SynopsisDirect difference ir spectra are presented as a function of hydration for lysozyme and u-lactalbumin, and detailed sequential hydration molecular events identified. Despite the strong sequence homology between the two proteins, and their expected conformational similarity, the hydration behavior of the polar groups is different for the two proteins. Using a Hill-type analysis, we conclude that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (a-lactalbumin) surface chemistry. The polar group hydration shows a clear cooperativity, which is quantitatively different in the two proteins. Complementary work suggests this cooperativity relates to a hydration-induced "loosening up" of the lysozyme conformation a t about 55 mol water/mol protein. u-Lactalbumin appears to "open up" more easily for hydration than does lysozyme, consistent with its lower stability against thermal and acid denaturation.

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Photoaffinity Labeling of Escherichia coli Ribosomes: New Approaches and Results

Cooperman¹,

Hall²,

Kerlavage³

et al. 1986

Springer Series in Molecular Biology

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The mechanism of chloramphenicol-induced changes in the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin. Evidence for puromycin and chloramphenicol sites on the 30S subunit

Cited by 19 publications

References 16 publications

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

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

Sequential hydration of dry proteins: A direct difference IR investigation of sequence homologs lysozyme and α‐lactalbumin

Photoaffinity Labeling of Escherichia coli Ribosomes: New Approaches and Results

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