Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA

Wybutinep(Ywye) , situated next to the 3′-side of the anti-codon of tRNAPhe from <unl>Saccharomyces</unl> <unl>cerevisiae</unl>, can be photo-crosslinked to mRNA when bound to <unl>Escherichia</unl> <unl>coli</unl> ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)<inf>6</inf>. In order to identify the site of reaction on the mRNA,5′-[32P]-labelledh pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be cross-linked to the U at the 5′-position of the corresponding UUU-co-don.