Photoaffinity labeling of <i>Escherichia coli</i> ribosomes with an aryl azide analogue of puromycin

Nicholson, Allen W.; Cooperman, Barry S.

doi:10.1016/0014-5793(78)80369-x

Cited by 29 publications

(20 citation statements)

References 29 publications

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“…7). The proximity of L27 to the peptidyl transferase center was also supported by affinity labeling studies with inhibitors of peptidyl transferase activity, such as chloramphenicol, carbomycin, tylosin, and spiramycin (8 -11), as well as with puromycin (12), an antibiotic that mimics the aminoacyl-adenosine moiety of aminoacyl-tRNA and is a substrate for peptidyl transferase (13). Direct evidence for the presence of L27 at the peptidyl transferase center was obtained through the use of derivatives of tRNA Phe containing photoreactive azidonucleotides within the 3Ј-terminal ACCA OH sequence (14,15).…”

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confidence: 79%

Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction

Wower

Zimmermann

1998

Journal of Biological Chemistry

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Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both coldand temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNA Phe to the A site, although the interaction of N-acetyl-Phe-tRNA Phe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.Protein L27 is one of the smallest and the most basic polypeptides in the Escherichia coli ribosome (1). A combination of immune electron microscopy and protein-protein crosslinking results places L27 at the base of the central protuberance on the interface side of the 50 S subunit (2). According to in vitro studies, L27 is a late assembly protein (3) and does not have an identifiable binding site on the 23 S rRNA. Chemical and UV cross-linking studies, however, demonstrated that L27 is associated closely with domain V of the 23 S rRNA (4 -6), a region that comprises part of the peptidyl transferase center (for review, see Ref. 7). The proximity of L27 to the peptidyl transferase center was also supported by affinity labeling studies with inhibitors of peptidyl transferase activity, such as chloramphenicol, carbomycin, tylosin, and spiramycin (8 -11), as well as with puromycin (12), an antibiotic that mimics the aminoacyl-adenosine moiety of aminoacyl-tRNA and is a substrate for peptidyl transferase (13). Direct evidence for the presence of L27 at the peptidyl transferase center was obtained through the use of derivatives of tRNA Phe containing photoreactive azidonucleotides within the 3Ј-terminal ACCA OH sequence (14,15). When bound to the ribosomal A or P site and irradiated, these probes became cross-linked predominantly to protein L27 and domain V of 23 S rRNA (15, 16).The contribution of protein L27 to the peptidyl transferase center has been addressed by a number of studies. It has been found, for instance, that the omission of L27 during in vitro reconstituti...

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confidence: 79%

Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction

Wower

Zimmermann

1998

Journal of Biological Chemistry

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“…[8-3H]Puromycin was prepared from [8-3H]puromycin aminonucleoside (Amersham) essentially as described for the preparation of [ 8-3H] -p-azidopuromycin (Nicholson & Cooperman, 1978), except that all products were ethanol-soluble and were purified by RP-HPLC (Kerlavage et al, 1985).…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

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

Kerlavage¹,

Cooperman²

1986

Biochemistry

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In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.

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“…There is less information available on protein L17. However, it crosslinks to a photo-affinity analogue of puromycin [20] and is highly stimulating in restoring peptidyl transferase activity [21 ].…”

Section: Resultsmentioning

confidence: 99%

A quaternary complex consisting of two molecules of tRNA and ribosomal proteins L2 and L17

et al. 1981

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Photoaffinity labeling of Escherichia coli ribosomes with an aryl azide analogue of puromycin

Cited by 29 publications

References 29 publications

Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction

Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction

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

A quaternary complex consisting of two molecules of tRNA and ribosomal proteins L2 and L17

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