Photohydration of uridine in the RNA of coliphage R17. I. Reductive assay for uridine photohydration.

Photohydration of uridine in the RNA of E. coli bacteriophage R17 has been investigated with the help of a new chemical method which avoids digestion of the irradiated polymer. The uridine photohydrates are reductively cleaved with sodium borotritiide and the radioactively labeled fragment, 1,3-propane-diol-(3)H(alpha), (3)H(alpha), (3)H(gamma), formed in this reaction is isolated and used as a measure for the extent of uridine photohydration in the irradiated RNA. The influence of the conformational state of R17-RNA on the cross section of uridine photohydration (sigma(H)) was investigated. The cross section for irradiation at 280 mmu at 25 degrees was largest in 6 M urea (sigma(H) = 0.053), slightly smaller in low salt (<10(-3)M Na(+); sigma(H) = 0.050), and substantially smaller in 0.15 M Na(+) (sigma(H) = 0.038). The closeness of the values for sigma(H) in urea and low salt indicates that a majority of the uridine residues in R17-RNA in the low salt medium at 25 degrees are not hydrogen bonded and destacked. The significant suppression of photohydration in 0.15 M Na(+) on the other hand shows that a substantial portion of the uridine residues in R17-RNA are participating in base pairing and base stacking under these conditions. Our results demonstrate that studies of uridine photohydration may, beyond their importance to photobiology, yield information specifically about the conformational state of the uridine-rich regions of a RNA, information which cannot be readily obtained by other techniques.