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.

Native calf thymus DNA was reacted with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) and its 7-fluoro and 7-iodo derivatives. Different ways of purification of the fluorene modified DNA samples were checked in order ot obtain a nucleic acid free from all noncovalently bound fluorene residues. The decrease in melting temperature in DNA samples modified by N-AcO-AAF(DNA-AAF) was carefully reinvestigated. From these experiments, we conclude that the melting temperature decrease is equal to 1.15 degree C per percent of modified bases, in DNA-AAF samples. Electric dichroism measurements on sonicated DNA samples modified by the different fluorene derivatives show the fluorene ring perpendicular to the helix axis in the case of the N-AcO-AAF and its fluoro derivative, and lying alone the phosphate-sugar backbone in the case of the iodo derivative. The results presented in this paper, along with those obtained earlier, led us to propose an "insertion-denaturation model" for the mode of binding of N-Aco-AAF and its fluoro derivative, and an "outside binding model" for the iodo derivative. Discrepancies with the data obtained by Chang et al.((1974) Biochemistry 13,2142-2148) concerning the melting temperature decrease and the electric dichroism results are observed and discussed.

A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.