PMID:9430711
| Citation |
Dey, D, Bochkariov, DE, Jokhadze, GG and Traut, RR (1998) Cross-linking of selected residues in the N- and C-terminal domains of Escherichia coli protein L7/L12 to other ribosomal proteins and the effect of elongation factor Tu. J. Biol. Chem. 273:1670-6 |
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| Abstract |
Five different variants of protein L7/L12, each with a single cysteine substitution at a selected site, were produced, modified with 125I-N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propion amide, a radiolabeled, sulfhydryl-specific, heterobifunctional, cleavable photocross-linking reagent that transfers radiolabel to the target molecule upon reduction of the disulfide bond. The proteins were reconstituted with core particles depleted of wild type L7/L12 to yield 70 S ribosomes. Cross-linked molecules were identified and quantified by the radiolabel. No cross-linking of RNA was detected. Two sites in the dimeric N-terminal domain, Cys-12 and Cys-33, cross-linked strongly to L10 and in lower yield to L11 but to no other proteins. The three sites in the globular C-terminal domain all cross-linked strongly to L11 and, in lower yield, to L10. Weaker cross-linking to 50 S proteins L2 and L5 occurred from all three C-terminal domain locations. The 30 S ribosomal proteins S2, S3, S7, S14, S18 were also cross-linked from all three of these sites. Binding of the ternary complex [14C]Phe-tRNA-elongation factor Tu.guanyl-5'-yl imidodiphosphate) but not [14C]Phe-tRNA.elongation factor Tu.GDP.kirromycin increased labeling of L2, L5, and all of the 30 S proteins. These results imply the flexibility of L7/L12 and the transient proximity of three surfaces of the C-terminal domain with the base of the stalk, the peptidyl transferase domain, and the head of the 30 S subunit. |
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| Keywords |
Amino Acid Substitution; Binding Sites; Centrifugation, Density Gradient; Crystallography, X-Ray; Cysteine/metabolism; Escherichia coli; Escherichia coli Proteins; Guanosine Diphosphate/metabolism; Models, Molecular; Mutagenesis, Site-Directed; Peptide Elongation Factor Tu/pharmacology; Protein Conformation; Pyridones/metabolism; Ribosomal Proteins/chemistry; Ribosomal Proteins/genetics; Ribosomal Proteins/metabolism; Structure-Activity Relationship |
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