Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine

Abstract

Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU rRNA from Thermotoga maritima (optimal growth 80 degrees C), one of the most deeply branched organisms in the Eubacterial phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA, occupying a net of approximately 14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and 11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (Mr 330.117), and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii.

FIGURE 1.

HPLC separation of RNase T1 digestion products of T. maritima 16S rRNA, annotated to show modified oligonucleotides determined from mass shifts in conjunction with data in Table 1. UV absorbance detection at 260 nm.

FIGURE 2.

Product ion mass spectrum from RNase T1 fragment M _r 2318, showing m³U-1498 adjacent to Am-1499. For clarity, only peaks used for sequence assignment are annotated. Sequence coverage from the four sequence ion series (a–B, d–H₂O, w, and y) (McLuckey et al. 1992) is shown by horizontal bars.

FIGURE 3.

Demonstration of presence of modified nucleoside N-330 in a total nucleoside digest of H. volcanii 16S rRNA (from previously acquired data) (Kowalak et al. 2000), detected from reconstructed ion chromatograms (RIC) for characteristic positively charged ions. (A) UV absorbance at 260 nm. (B) RIC for the MH⁺ ion of N-330 (m/z 331). (C) RIC for the protonated base fragment ion of N-330 (m/z 199).