Abstract

RNA can be radiolabeled in vitro with 125I in uridine and cytidine residues by initial mercuration of these pyrimidine residues by the method of Dale et al. [Dale, R. M. K., Martin, E., Livingston, D. C., & Ward, D. C. (1975) Biochemistry 14, 2447-2457] and subsequent electrophilic displacement of the mercury with 125I+ generated by the method of Commerford [Commerford, S. L. (1971) Biochemistry 10, 1993-2000]. These two reactions can be manipulated to produce intact high specific activity 125I-labeled mRNA containing approximately equal specific activity in their 5-[125I]iodouridine and 5-[125I]iodocytidine residues. In vitro radiolabeling of satellite tobacco necrosis virus RNA (STNV RNA) by this procedure yields a 125I-labeled mRNA that is biologically active in ribosome protection analyses in that one obtains the correct translation initiation fragments of the mRNA. Exhaustive digestion of a specific 125I-labeled 32 nucleotide long initiation fragment of STNV RNA using four separate nucleotide-specific digestion (hydrolysis) reactions yields four different populations of 125I-labeled digestion products that can be resolved by two-dimensional fingerprint procedures. Characterization of all these 125I-labeled digestion products followed by overlap nucleotide sequence comparisons of these specific digestion products allows a nucleotide sequence determination of the original 32 nucleotide long translation initiation fragment of this mRNA. This suggests that this overall in vitro procedure can be used to determine the nucleotide sequence of the translation initiation site(s) of any mRNA.