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.
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.
Dey
D
D
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616, USA.
Bochkariov
D E
DE
Jokhadze
G G
GG
Traut
R R
RR
D019943:Amino Acid Substitution; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000494:pharmacology*; D011487:Protein Conformation; D011728:Pyridones / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D011728:Pyridones; D012269:Ribosomal Proteins; C512844:rplL protein, E coli; D006153:Guanosine Diphosphate; C016700:ribosomal protein L7-L12; D010444:Peptide Elongation Factor Tu; D003545:Cysteine; C008536:mocimycin