DISTRIBUTION OF CROSS-LINKS BETWEEN MESSENGER-RNA ANALOGS AND 16-S RIBOSOMAL-RNA IN ESCHERICHIA-COLI 70-S RIBOSOMES MADE UNDER EQUILIBRIUM CONDITIONS AND THEIR RESPONSE TO TRANSFER-RNA BINDING

The interaction between mRNA and Escherichia coli ribosomes has been studied by photochemical crosslinking using mRNA analogues that contain 4-thiouridine (s(4)U) or s(4)U modified with azidophenylacyl bromide (APAB), either two nucleotides upstream or eight nucleotides downstream from the nucleotide sequence ACC, the codon for tRNA(Thr). The sequences of the mRNA analogues were described earlier (Stade, K, Rinke-Appel, J., and Brimacombe, R (1989) Nucleic Acids Res. 17, 9889-9908; Rinke-Appel, J., Stade, K., and Brimacombe, R. (1991) EMBO J, 10, 2195-2202), Under equilibrium conditions, both of these mRNA analogues bind and cross-link to 70 S ribosomes without the presence of tRNA(Thr); however, there are significant increases both in binding and particularly in cross-linking in the presence of the tRNA(Thr). Four regions contain cross-linking sites that increase in the presence of tRNA, C-1395, A(532) A(1196) (and minor sites around these three positions), and C-1539/U-1532. Three other cross-linking sites, U-723, A(845), and U-1381, show very little change in extent of crosslinking when tRNA is present. A conformational change in the 30 S subunit allowing additional accessibility to the 16 S rRNA by the mRNA analogues upon tRNA binding best explains the behavior of the tRNA-dependent and tRNA-independent mRNA-16 S rRNA cross-linking sites.