Implications of a functional large ribosomal RNA with only three modified nucleotides.

The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, 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 analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.