A Domain of 23S Ribosomal RNA in Search of a Function
The 23S-like ribosomal RNAs (rRNA) consist of six main structural domains that are stabilized by long range base pairing interactions. Domain IV has remained one of the most highly conserved of these during evolution, and this implies that it plays a crucial role at some stage of protein biosynthesis. In this article, we examine the structure of domain IV of Escherichia coli, 23S RNA experimentally and consider its possible functional roles.
We probed the accessible adenosines, and some cytidines, in this domain using diethyl pyrocarbonate and dimethyl sulphate/hydrazine, respectively, and we isolated homogeneous RNA fragments, for analysis, using a DNA hybridization technique (Van Stolk, B. and Noller, H.F. J. Mol. Biol., 180, 151, 1984). The results were compared with published secondary structural models that are based primarily on phylogenetic sequence comparisons. Although these models still contain uncertainties, the data were generally in good agreement with recently revised versions. Most of the reactive adenosines and cytidines were concentrated in the putative loop and interhelical regions. Only four moderately reactive adenosines, and no cytidines, were found in putative double helices under “native” conditions (10 mM magnesium). Two of these occurred in the putative helix 10 that appears to be disproven both experimentally and by phylogenetic criteria. The experimental data were compatible, however, with other helical segments, for which there is no phylogenetic support because the region has been either too conserved or too labile during evolution. Under semi-denatured conditions (1 mM EDTA) increased reactivity was confined to relatively few nucleotides, situated mainly in loop and interhelical regions, which suggests that the whole domain exhibits a stable and intricate tertiary structure. This presumption is supported further by the low reactivity of several nucleotides in “unstructured” regions under both native and semi-denatured conditions.
Three putative functional sites were identified on the basis of their highly conserved sequences, post transcriptional modifications, intron splicing sites, and location on the ribosomal surface. Possible functions for each of these sites are considered and evaluated.
KeywordsDomain Versus Terminal Loop Peptidyl Transferase Secondary Structure Model Peptidyl Transfer
Unable to display preview. Download preview PDF.
- Garrett, R.A., 1983, Structure and role of eubacterial ribosomal proteins, in: “Genes: Structure and Expression”, A. Kroon, ed. J. Wiley and Sons, London.Google Scholar
- Garrett, R.A., Vester, B., Leffers, H., Sørensen, P.M., Kjems, J., Olesen, S.O., Christensen, A., Christiansen, J. and Douthwaite, S., 1984, Mechanisms of protein-RNA recognition and assembly in ribosomes, in: “Gene Expression”, ed. Clark, B.F.C. and Petersen, H.U., Munksgaard, Copenhagen, 1984.Google Scholar
- Kjems, J. and Garrett, R.A., 1985, An intron in the 23S rRNA gene of the archaebacterium Desulfurococcus mobilis, Nature, in press.Google Scholar
- Leffers, H., Kjems, J. and Garrett, R.A., 1985, The gene sequences of the 23S RNAs of Halococcus morrhuae and Desulfurococcus mobilis: A comparative study, manuscript in preparation.Google Scholar
- Seilhamer, J.J., Gutell, R.R., Cummings, D.J., 1984, Paramecium mitochondrial genes. II. Large subunit rRNA gene sequence and microevolu-tion, J. Biol. Chem. 259:5167.Google Scholar
- Sloof, P., Van Den Burg, J., Voogd, A., Benne, R., Agostinelli, M., Borst P., Gutell, R. and Noller, H.F., 1985. Further characterization of the extremely small rRNAs from trypanosomes: A detailed comparison of the 9S and 12S RNAs from Crithidia fasciculata and Trypanosoma brucei with rRNAs from other organisms, Nucl. Acids Res., 13:4171.CrossRefGoogle Scholar
- Zagorska, L., Van Duin, J., Noller, H.F., Pace, B., Johnson, K.D. and Pace, N.R., 1984, The conserved 5S rRNA complement to tRNA is not required for translation of natural mRNA, J. Biol. Chem. 259:2798.Google Scholar