THE C-TERMINAL DOMAIN OF ESCHERICHIA-COLI RIBOSOMAL-PROTEIN L7/L12 CAN OCCUPY A LOCATION NEAR THE FACTOR-BINDING DOMAIN OF THE 50S SUBUNIT AS SHOWN BY CROSS-LINKING WITH N-[4-(PARA-AZIDOSALICYLAMIDO)BUTYL]-3-(2'-PYRIDYLDITHIO)PROPIONAMIDE

All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis. The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propionamide. Properties of this reagent are described. It was first radiolabeled with I-125 and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89. Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89. Irradiation of there constituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components. Reductive cleavage of the disulfide cross-link resulted in transfer of the I-125 label from L7/L12Cys89 to the other cross-linked components. Two radiolabeled proteins were identified, L11 and L10. The location of both of these proteins is well established to be at the base of the L7/L 1 2 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors. The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain. The cross-linking method with radiolabeled N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.