RECOMBINANT PHOTOREACTIVE TRANSFER-RNA MOLECULES AS PROBES FOR CROSS-LINKING STUDIES

Photoreactive tRNA derivatives have been used extensively for investig ating the interaction of tRNA molecules with their ligands and substra tes. Recombinant RNA technology facilitates the construction of such t RNA probes through site-specific incorporation of photoreactive nucleo sides. The general strategy involves preparation of suitable tRNA frag ments and their ligation either to a photoreactive nucleotide or to ea ch other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The a pplication of this methodology to the study of tRNA binding sites on t he ribosome is discussed, and a model of the tRNA-ribosome complex is presented.