Small‐Angle X‐Ray Titration Study on the Complex Formation between 5‐S RNA and the L18 Protein of the Escherichia coli 50‐S Ribosome Particle

Abstract

The 5-S RNA (A) and the L18 protein (B) from Escherichia coli ribosomes form one single AB complex in the concentration ranges supposed to prevail in vivo; at concentrations of L18 higher than 40 mM there is some indication for a minor species, most probably an AB2 species. This is indicated from the X-ray scattering titration data of the 5-S RNA/Ll8 system recorded at 21 "C in ribosomal reconstitution buffer. As a result of the 1 : 1 complex formation, there is a relatively small but defined increase in the radius of gyration from 3.61 to 3.85 nm. This result as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L18 model is quite far from the electron density centre and where protein L18 interacts with one or both of the minor arms of the supposed Y-shaped 5-S RNA molecule.The interactions between a nucleic acid and a protein molecule are of great general interest in molecular biology, since they are involved both in the transcription and the translation processes. Transfer RNA (tRNA) and 5-S RNA are two examples of nucleic acids that specifically interact with proteins ; and they are of special interest since their relatively small sizes, 75 and 120 nucleotides, respectively, make possible structural studies in some detail. The complex formation between an aminoacyl-tRNA ligase and its tRNA substrate was recently investigated using small-angle X-ray scattering titration, a method especially developed for studying macromolecular interaction [I]; the result indicated that the relatively large ligase molecule ( M , 114 000) dimerized at the complex formation and partly engulfed the smaller tRNA molecule ( M , 25000) [2]. Within the Escherichia coli ribosome, on the other hand, the protein/nucleic acid interactions seem to be quite different from those of the tRNA ligase systems, since here relatively small proteins react with the larger rRNA molecules. This is illustrated by the present study which involves complex formation between 5-S RNA of E. coli ( M , 39000 [3]) and the protein L18 ( M , 12770 [4]), c$ [6]. As a prerequisite for this study the protein L18 has been characterized by the small-angle X-ray scattering method [5], and 5-S RNA has been the subject of a comprehensive study where a tertiary 5-S RNA model was described [3]. The present study, which suggests an idea of how 5-S RNA and L18 interact, deals with both equilibrium data and structural data for the complexes of 5-S RNA with protein L18. MATERIALS AND METHODS MaterialsThe 5-S RNA was prepared from E. coli ribosomes (strain A19) using the method described by Monier and Feunteun [7]. The final product migrated as a single band in polyacrylamide gel electrophoresis, and the distance moved corresponded to native 5-S RNA [8]. As revealed by both X-ray scattering data and gel electrophoresis [9], the 5-S RNA samples were biologically active in the sense that they formed specific complexes with the ribosomal proteins L18 and L25.The protein L18 was prepared from 50-S subunits of E. coli A19....

Structure and macromolecular interactions of 5S RNA. 2. Parameters for the interaction of ribosomal proteins L5, L18, and L25 with 5S RNA from Escherichia coli

“…All three complexes were previously shown to contain about one molecule of protein per molecule of RNA at saturation , and we found no evidence to support the earlier suggestion that the molar ratio of LI8 to 5S RNA reaches 2:1 (Feunteun et al, 1975). Our estimate of approximately 10s M_l for the ÁV of the L18-5S RNA interaction indicates a much greater stability than do previous values of 107 and 106 M~1 obtained by fluorescence and X-ray scattering techniques, respectively (Feunteun et al, 1975;Ósterberg & Garrett, 1977). Reasons for these discrepancies are unknown, although the various procedures employed are quite different in principle.…”

“…Low-angle X-ray scattering studies suggest that both protein and RNA are highly elongated (Ósterberg et al, 1976a,b). The positively charged N terminus of LI8 may therefore be accessible for binding to the 5S RNA (Ósterberg & Garrett, 1977) much as has been suggested for the association of histone with DNA (Weintraub & van Lente, 1974) where rather similar changes in CD have been observed (Adler et al, 1975). Although recent evidence suggests that the Nterminal residues of L18 may not be essential for L18-5S RNA association (Newberry et ah, 1978), electrostatic interactions between basic amino acid side chains and negatively charged phosphate groups would undoubtedly contribute additional stability to the complex and are compatible with the substantial binding entropies involved.…”

Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

“…This incomplete processing resulted from chloramphenicol binding (Jordan et al, 1971) (Osterberg and Garrett, 1977) and 107/M from a fluorescence study (Feunteun et al, 1975). Each of these results lies well below the estimate of 2 x 108/M from a filter binding assay (Spierer et al, 1978).…”

Structure and Function of the Acidic Ribosomal Stalk Proteins

“…Other rproteins, e.g. L18 and L25, have been ascribed an elongated shape based on similar solution studies [127,138] but subsequently NMR and crystal structures of the proteins alone and in complexes [10,139,140] showed globular molecules. In the special case of L12, elongation and compaction may interchange during the translational cycle.…”