LOCATION AND DOMAIN-STRUCTURE OF ESCHERICHIA-COLI RIBOSOMAL-PROTEIN L7 L12 - SITE-SPECIFIC CYSTEINE CROSS-LINKING AND ATTACHMENT OF FLUORESCENT-PROBES/

Five different variants of L7/L12 containing single cysteine substitut ions, two in the N-terminal (NTD) and three in the C-terminal domain ( CTD), were produced, modified with 4-(p-azidosalicylamido)butyl]-3-(2' -pyridyldithio) propionamide ([I-125]APDP), a sulfhydryl-specific, het erobifunctional, cleavable photo-cross-linking reagent, and reconstitu ted into ribosomes. These were irradiated, the total proteins were ext racted and reductively cleaved, and the cross-linked proteins were ide ntified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used t o attach a rhodamine dye. The formation of ground-state rhodamine dime rs caused the appearance of a new absorption band at 518 nm that was u sed to estimate the extent of interaction of the probes in the free pr otein and in complexes with L10. The three sites in the CTD, but not t he N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Bi nding to the ribosome and, therefore, function were blocked by zero-le ngth cross-linking within the NTD, but not the CTD. Binding also disru pted rhodamine dimers in the NTD. No rhodamine dimers formed in the CT D.