Topography of 5.8 S rRNA in rat liver ribosomes. Identification of diethyl pyrocarbonate-reactive sites (original) (raw)

1982, Journal of Biological Chemistry

The topography of 5.8 S rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 5-28 S rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gelsequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the ttAU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two-G-A-A-C-sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes. Although our knowledge of the structure of ribosomal RNAs is growing rapidly, the elucidation of their functional roles in protein synthesis continues to be a formidable problem. A variety of studies indicate that the 3'-end of the single 16-18 S RNA component in the small ribosomal subunit is probably involved in mRNA binding (1) and several functional roles also have been suggested for the 5 S rRNA component including tRNA binding (2), a GTPase activity (3), and subunit interaction (4,5). There is little solid evidence to support any of these roles and it seems unlikely that all of these roles reside in this one small RNA molecule. Virtually nothing is currently known about the function of the 23-28 S rRNA component of the large ribosomal subunit or the eukaryotic 5.8 S rRNA. Recently, however, several lines of speculation have suggested that the 5.8 S RNA molecule may replace in part the 5 S RNA in its tRNA-binding role (6, 7). The 5.8 S rRNA, which is hydrogen-bonded to its cognate * This work was supported by Grant A6666 of the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.