| Summary: | (1) N-Acetyl{('3)H}lysyl-tRNA('Lys) and N-acetyl{('3)H}glutamyl-tRNA(,2)('Glu) were cross-linked to the P site of Escherichia coli 70S tight-couple ribosomes by irradiation with light of 300 to 400 nm. Covalent attachment was dependent on the presence of polynucleotide message and nearly 85% of the ('3)H-labeled amino acids could be released from the cross-linked complexes by puromycin. Cross-linking was stimulated in the presence of acetone and reversed by subsequent exposure to light of 254 nm. Centrifugation of covalent complexes through sucrose gradients revealed that tRNA was attached only to 30S subunits, and further analysis demonstrated that linkage was to 16S RNA, but not to ribosomal proteins. Partial RNase T(,1) hydrolysis of N-acetylaminoacyl-tRNA--16S RNA complexes allowed localization of the site of cross-linking to the 8S RNA fragment derived from the 3' 40% of the 16S RNA. When N-acetyl-aminoacyl-tRNA--8S RNA complexes were electrophoresed in polyacrylamide gels under denaturing conditions, a major fraction of the tRNA was found to be cross-linked to one or more rRNA subfragments of 100-125 nucleotides. (2) N-Acetylvalyl-tRNA(,1)('Val) was bound to the P site of uniformly ('32)P-labeled 70S tight-couple ribosomes and cross-linked to 16S RNA in the 30S subunit by irradiation with near ultraviolet light. Following partial RNase T(,1) digestion of N-acetylvalyl-tRNA(,1)('Val)--16S{('32)P}RNA complexes, the rRNA fragments to which the tRNA cross-links were isolated by two-dimensional gel electrophoresis according to the diagonal method. After the first dimension, the tRNA-rRNA cross-link was cleaved by photolysis at 254 nm. Upon electrophoresis in the second dimension, RNA segments previously attached to tRNA migrated to positions beneath the diagonal. The appearance of oligonucleotides below the diagonal was dependent on the presence of tRNA in the initial reaction mixture and required photoreversal of the cross-link. Sequence analysis revealed that the smallest of three nested rRNA fragments containing the site of tRNA attachment encompassed residues 1362 through 1497 of the 16S RNA. Following complete RNase T(,1) hydrolysis of N-acetylvalyl-tRNA(,1)('Val)--16S (('32)P)RNA complexes, the cross-linked tRNA-rRNA oligonucleotide was isolated by polyacrylamide gel electrophoresis. The cross-linked adduct contained a ('32)P-labeled nonanucleotide from the 16S RNA and an unlabeled pentadecanucleotide from the tRNA. The sequence of the nonanucleotide was determined to be U-A-C-A-C-A-C-C-G, which occupies positions 1393-1401 of the 16S RNA and is located within an evolutionarily conserved region. Secondary digestion analysis of the covalent tRNA-rRNA oligonucleotide revealed that the cross-linked residue in the 16S RNA was C(,1400). The site of cross-linking in the tRNA was ascertained using N-acetylvalyl-tRNA(,1)('Val)--16S RNA complexes prepared from nonradioactive ribosomes. After complete RNase T(,1) digestion of these complexes, the RNA was labeled at the 3' end with {5'-('32)P}pCp. The covalent tRNA-rRNA oligonucleotide isolated from the mixture released a single end-labeled component upon photoreversal of the cross-link. Chemical sequence analysis demonstrated that this product was the anticodon-containing pentadecanucleotide of tRNA(,1)('Val), C-A-C-C-U-C-C-C-U-cmo('5)U-A-C-m('6)A-A-G(,39), and that the site of covalent attachment to 16S RNA was the 5' anticodon nucleotide, cmo('5)U(,34). The structure of the cross-linked adduct is proposed to be a cyclobutane dimer between cmo('5)U(,34) of tRNA(,1)('Val) and C(,1400) of the 16S RNA.
|
|---|
