A protonated base pair participating in rRNA tertiary structural interactions

In the recently published X-ray crystallographic structure for the 50S subunit of Haloarcula marismortui ribosomes, residue U2546 of the 23S rRNA forms a non-Watson-Crick base pair with U2610. The corresponding residues in the secondary structure of the Escherichia coli 23S molecule are U2511 and C2575, and it follows that the latter base (C2575) should be protonated in order to form a base pair that is isostructural with its counterpart in H.marismortui. This prediction was demonstrated experimentally by reduction with sodium borohydride followed by primer extension analysis; borohydride is able to reduce positively charged bases, yielding products which block reverse transcription. In the course of the analysis a further charged base pair (AH(+)1528-G1543) was identified in the E.coli 23S molecule. Both charged pairs (U2511-CH(+)2575 and AH(+)1528-G1543) were only observed in the context of the intact ribosomal subunit and were not seen in deproteinized rRNA.