COVALENT CROSS-LINKING OF TRANSFER RIBONUCLEIC-ACID TO THE RIBOSOMAL-P SITE - SITE OF REACTION IN 16S RIBONUCLEIC-ACID

N-Acetyl[3H]aminoacyl derivatives of tRNA1val and tRNA1,Ser from Escherichia coli, and of tRNAVal from BacIIIus subtilis, have been covalently cross-linked to the P site of 70S tight-couple ribosomes by irradiation with 310-350-nm light. The irradiated ribosomes were dissociated into 30S and 50S subunits, and ribosomal RNA was extracted from each of the particles with phenol. Sucrose gradient analysis of this material revealed that the covalently bound N-acetyl[3H]aminoacyl-tRNA cosedimented with 16S ribosomal RNA in all three cases. Following limited digestion of the N-acetyl[3H]aminoacyl-tRNA-16S RNA adducts with RNase T1 the tRNA moiety remained attached to a fragment of about 8 S which encompasses the entire 3' third of the 16S RNA with the exception of approximately 50 residues at the 3' terminus. Polyacrylamide gel electrophoresis in 6 M urea was used to separate the 8S fragment into a number of smaller components through the exposure of “hidden breaks” introduced during RNase treatment. Under these conditions, most of the 3H radioactivity from the 8S fragment complex migrated in a band which consisted of 150-200 nucleotides and was larger than any of the major subfragments arising from non-cross-linked 8S RNA. Assuming the tRNA to be intact, the main 3H-labeled band should contain a sequence of 75-125 bases derived from the 16S RNA. Since the electrophoretic mobility of this component was not altered by prior incubation with proteolytic enzymes, it is unlikely that the cross-link is mediated by protein. On the basis of these results, we suggest that a short segment of the 16S RNA, located near the 3' end but not within 50 bases of the 3' terminus, comprises part of the ribosomal P site and is juxtaposed to the anticodon of the peptidyl-tRNA in such a way that direct photochemical cross-linking of nucleotides in the two molecules can occur. © 1979, American Chemical Society. All rights reserved.