Intramolecular and intermolecular snRNA cross-links were generated by irradiating HeLa nuclear extract with 365 nm light in the presence of the psoralen derivative AMT. After deproteinization, cross-linked RNAs were resolved by gel electrophoresis and identified as anomalously migrating species by Northern blotting. In addition to the U4/U6 snRNA cross-link, we detected an intermolecular U2/U6 cross-link, as well as several apparently intramolecular Ul, U2, and U5 cross-links. Photoreversal of the U2/U6 cross-link with 254 nm irradiation released stoichiometric amounts of U2 and U6 snRNA. To localize the U2/U6 cross-link, the 3' end of U2 in the purified U2/U6 complex was labeled selectively using a novel oligonucleotide "splint" technique. The labeled U2/U6 complex was then subjected to rapid enzymatic RNA sequencing or to targeted digestion of the U2 and U6 components of the complex by RNase H and a panel of complementary oligonucleotides. The U2/U6 cross-link is located upstream of nucleotide 15 in U2 and downstream of nucleotide 85 in U6, suggesting that the phylogenetically conserved base-pairing between these regions (6 consecutive base pairs in human, Drosophila melanogaster, and Caenorhabditis elegans, 7 in Schizosaccharomyces pombe and Jiypanasoma brucei, 8 in Pisutn sativum, 11 in Saccharomyces cerevisiae) is significant.

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The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained.