Nucleic Acids Research, 1989, Vol. 17, No. 21 8767-8782
© 1989
CHEMISTRY |
Ribosomal proteins S7 and L1 are located close to the decoding site of E.coli ribosome — affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3'-end of the anticodon
ski
Institute of Bioorganic Chemistry, Polish Academy of Sciences Noskowskiego 12/14, 61704 Pozna, Poland
*To whom correspondence should be addressed
Received August 21, 1989. Accepted September 28, 1989.
Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16–17 and 23–24å, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A, U, G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 16S rRNA reaches significant levels only in the absence of the message.