Primary and secondary structures of Escherichia coli MRE 600 23S ribosomal RNA. Comparison with models of secondary structure for maize chloroplast 23S rRNA and for large portions of mouse and human 16S mitochondrial rRNAs

Abstract

We determined 90% of the primary structure of E.coli MRE 600 23S rRNA by applying the sequencing gel technique to products of T1, S1, A and Naja oxiana nuclease digestion. Eight cistron heterogeneities were detected, as well as 16 differences with the published sequence of a 23S rRNA gene of an E.coli K12 strain. The positions of 13 post-transcriptionally modified nucleotides and of single-stranded, double-stranded and subunit surface regions of E.coli 23S rRNA were identified. Using these experimental results and by comparing the sequences of E.coli 23S rRNA, maize chloro. 23S rRNA and mouse and human mit 16S rRNAs, we built models of secondary structure for the two 23S rRNAs and for large portions of the two mit rRNAs. The structures proposed for maize chloroplast and E.coli 23S rRNAs are very similar, consisting of 7 domains closed by long-range base-pairings. In the mitochondrial 16S rRNAs, 3 of these domains are strongly reduced in size and have a very different primary structure compared to those of the 23S rRNAs. These domains were previously found to constitute a compact area in the E.coli 50S subunits. The conserved domains do not belong to this area and contain almost all the modified nucleotides. The most highly conserved domain, 2042-2625, is probably part of the ribosomal A site. Finally, our study strongly suggests that in cytoplasmic ribosomes the 3'-end of 5.8S rRNA is basepaired with the 5'-end of 26S rRNA. This confirms the idea that 5.8S RNA is the counterpart of the 5'-terminal region of prokaryotic 23S rRNA.

Structure and function of ribosomal RNA

“…The most extreme example is the ribosome of Crithidia fasciculata, whose large subunit contains no less than four extra small RNA species in addition to 5S and 5.8S RNA (Schnare et al, 1983), although it has yet to be established whether all of these have 23S RNA counterparts. In contrast to this type of extra processing event, it has recently been proposed that some fungal mitochondrial ribosomes (which, as noted above, lack 5S RNA) may have actually incorporated a 5S-like sequence into their large subunit RNA molecules Dron et al (1982) Brosius et al (1978, Carbon et al (1978 Carbon et al (1981) Kop et al (1984a) Iwami et al (1984) 12S Eperon et al (1983) 16S Van Etten et al (1980) 16S Saccone et al (1981) 16S Eperon et al (1980) 16S Anderson et al (1982) 20Sb Seilhammer & Cummings (1981) 20Sb Seilhammer et al (1984b) 21S Sor & Fukuhara (1983) 23S Kochel & Kuntzel (1982) 26S Dale et al (1984) 23SC Edwards & Kossel (1981) 23SC Takaiwa & Sugiura (1982) 23S Brosius et al (1980), Branlant et al (1981) 23S Kumano et al (1983, Douglas & Doolittle (1984) 23S Kop et al (1984b) 16S Gupta et al (1983) 16S Leffers & Garrett (1984) McCarroll et al (1983) Takaiwa et al (1984) Messing et al (1984) Rubtsov et al (1980 Hadjiolov et al (1984), Chan et al (1983) There are a small number of modified nucleotides in ribosomal RNA. In E. coli, the 16S rRNA contains nine methylated bases (Carbon et al, 1979) and the 23S rRNA ten methylated bases and three pseudouridine residues .…”

“…The main effort has concentrated on the E. coli 16S and 23S rRNA, and three essentially similar secondary structure models were proposed for both molecules (16S RNA: Stiegler et al, 198 1a;Zwieb et al, 1981;and 23S RNA: Glotz et al, 1981;Branlant et al, 1981). In each case the structures were derived by a two-track approach, involving firstly the collection of experimental data from the E. coli RNA and secondly phylogenetic comparisons with ribosomal RNA sequences from other organisms (reviewed by Brimacombe et al, 1983;Woese et al, 1983;Noller, 1984).…”

“…In the case of the large subunit, models have been published for mammalian mitochondrial 16S RNA (e.g. Glotz et al, 1981;Branlant et al, 1981) as well as for eukaryotic 26S RNA (Veldman et al, 1981) and 28S RNA (e.g. Michot et al, 1984;Hadjiolov et al, 1984).…”

Elongation factor G protects a nuclease‐sensitive site of 23 S RNA within the ribosome

“…One of these sites (site 1, see scheme in fig.1) is positioned at a distance of about 1171-l 178 nucleotides from the 5 '-end of 23 S RNA [31,32] and its hydrolysis gives two fragments detected by SDS-PAGE without denaturation of the RNA secondary structure: the 3 '-terminal 18 S and 5 '-terminal 13 S fragments . Electrophoresis of the hydrolyzate after preliminary denaturation of the RNA (5 min at 75-8O"C, 0.2% SDS [24] or 6.6-8.0 M urea [25,30]) reveals another specific cleavage site (site 2 in the scheme) at a distance of about 1000 nucleotides from the 3'-end of 23 S RNA with formation of the 3'-terminal 11 S [28,32) (or I2 S [30,33]) fragment.…”

The three-dimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment ofEscherichia coli50S ribosomal subunits withbis-(2-chloroethyl)-methylamine

“…The helix numbers within the secondary structure (cf. Figure 4) h. The T1-oligonucleotide ACCCUUUAAGp contains several sequence heterogeneities (20). (Table I), and also contains a characteristic methylated oligonucleotide (marked 'X' in Figure 2a) with the sequence mAAGp (ref.…”