IMPLICATIONS OF A FUNCTIONAL LARGE RIBOSOMAL-RNA WITH ONLY 3 MODIFIEDNUCLEOTIDES

The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucle otides could be important structural or functional elements in the cat alytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in v itro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose m ethyltransferase that specifically modifies a universally conserved nu cleotide, G2270. in the peptidyl transferase center of the mitochondri al large ribosomal RNA (21S). Since the loss of this modification in y east mitochondrial 21S rRNA severely affects the assembly of 54S subun its, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required far the assembly of 50S subunits. Gm could be a critical structural determinant for the co rrect folding of the rRNA, the binding of one or more ribosomal protei ns, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analys is using combined high performance liquid chromatography-mass spectrom etry, the modification status of the yeast mitochondrial rRNAs was ree xamined, revealing the presence of Gm, Um and pseudouridine (Psi) in 2 1S rRNA. The Um was mapped to nucleotide 2791, which corresponds to th e ribose methylated and universally conserved U2552 in E coli 23S rRNA , and the Psi has been recently mapped to position 2819, which corresp onds to Psi 2580 in E coli 23S rRNA. The retention of Um and Psi nucle otides in the peptidyl transferase center of the otherwise minimally m odified mitochondrial rRNAs suggests that these modifications, like Gm 2270, might be essential for ribosome assembly or function or both.