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Nucleic Acids Research, 1991, Vol. 19, No. 4 801-808
© 1991

MOLECULAR BIOLOGY

Neighbourhood of the central fold of the tRNA molecule bound to the E.coli ribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Jan Podkowinski and Piotr Gornicki*

Institute of Bioorganic Chemistry, Polish Academy of Sciences Noskowskiego 12/14, 61704 Poznan, Poland

* To whom correspondence should be addressed at Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637. USA

Received November 15, 1990. Revised January 24, 1991. Accepted January 24, 1991.

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E.coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10 – 11 Å and 18 – 19 Å, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poiy(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon Irradiation of the respective noncovalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product Is reproducibly found in a protein fraction prepared from the purified 50s subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2 – 4 fold in the presence of poly(A,U,G). The crosslinking sites are located between nucleotides 1302 and 1398 in 16s rRNA and between nucieotldes 2281 and 2358 in 23s rRNA.


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