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Nucleic Acids Research, 1984, Vol. 12, No. 21 8181-8191
© 1984

MOLECULAR BIOLOGY

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

Günter Steiner, Reinhard Lührmann+ and Ernst Kuechler*

+Max-Planck-Institut für Molekulare Genetik 1000 Berlin 33 (Dahlem), FRG Institut für Biochemie der Universität Wien 1090 Vienna, Währingerstrasse 17, Austria

*To whom correspondence should be addressed

Received September 24, 1984. Accepted October 16, 1984.

Wybutinep(Ywye) , situated next to the 3'-side of the anti-codon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. 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.


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