POSTTRANSCRIPTIONAL MODIFICATION OF THE CENTRAL LOOP OF DOMAIN-V IN ESCHERICHIA-COLI 23-S RIBOSOMAL-RNA

Knowledge of the sites, structures, and functional roles of posttransc riptional modification in rRNAs is limited, despite steadily accumulat ing evidence that rRNA plays a direct role in the peptidyl transferase reaction and that modified nucleotides are concentrated at the functi onal center of the ribosome. Using methods based on mass spectrometry, modifications have been mapped in Escherichia coli 23 S rRNA in the c entral loop of domain V, a region of established interaction between 2 3 S RNA and tRNA. Two segments of RNA were isolated following protecti on with oligodeoxynucleotides and nuclease digestion: residues 2423-24 73 (51-mer) and 2481-2519 (39-mer). Dihydrouridine was located at posi tion 2449, within the RNase T-1 hydrolysis product 2448-ADAACAGp-2454, as evidenced by a molecular mass 2 daltons higher than the gene seque nce-predicted mass. This nucleoside, which is nearly ubiquitous in tRN A (where it is involved in maintenance of loop structure), is two base s from A-2551, a previously determined site of interaction between 23 S RNA and the CCA-aminoacyl terminus of tRNA at the ribosomal P-site. The oligonucleotide 2496-CACmCUCGp-2502 was isolated and accurately ma ss measured, and its nucleoside constituents were characterized by hig h performance liquid chromatography-mass spectrometry; there was no ev idence of modification at position 2501 as implied by earlier work. Us ing similar techniques, the modified adenosine at position 2503 was un ambiguously determined to be 2-methyladenosine in the fragment 2503-m( 2)A Psi Gp-2505.