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.

Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E. coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis. The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA. Sequence comparison with the 23S rDNA from Z. mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species. The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species. Some ribosomal structural "'switches" were found, one involving 5S rRNA.

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The nucleotide sequence of 23S rDNA from Zea mays chloroplasts has been determined. Alignment with 23S rDNA from E.coli reveals 71 percent homology when maize 4.5S rDNA is included as an equivalent of the 3' end of E.coli 23S rDNA. Among the conserved sequences are sites for base modification. Chloramphenicol sensitivity and ribosomal subunit interaction. A proposal for the base pairs formed between 16S and 23S rRNAs during the 30S/50S subunit interaction is presented. The alignment of maize 23S rDNA with that of E.coli reveals three small insertion sequences of 25, 65 and 78 base pairs, whereas maize 16S rDNA shows only deletions when compared with the E.coli species.

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