The Composition and Properties of the Escherichia coli 5–S RNA–Protein Complex

Abstract

At a high concentration of MgC12 (30 mM) and a low concentration of proteins from the 50-S subunit (0.2 mgiml), only three proteins, L15, L18 and L25, bind to 5-S RNA in significant amounts. On the other hand, in a buffer containing only 1 mM Mg C12, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1 .5 mg/ml, a large, stable complex can form between immobilized 5-S RNA and 50-S ribosomal proteins. This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possesses properties relevant to the function of the 50-S subunit: it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 x M. The complex formed with 5-S RNA immobilized on an affinity column interacts also with 30-S subunits. The 5-S RNA-protein complex is interpreted as a sub-ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichiu coli ribosome.

Studies on the interactions of low molecular weight ribosomal RNAs and ribosomal proteins

“…Other groups have adopted the affinity chromatography methods developed in this laboratory to determine the ribosomal proteins which interact with low molecular weight ribosomal RNAs and tRNAs (Ulbrich et al, 1980a;Metspalu et al, 1982a). Ulbrich et al (1980a) showed that at room temperature (22°C), in binding buffer containing 0.3M KCl, proteins L5, L18 and L25 were bound to E. coli 5S RNA in agreement with the .results of our experiments.…”

“…When the ribosomal protein concentration was further increased to I,8mg/mL or binding buffer containing 1 mM MgCl2 and 200 mM KCl was used, there was an increase in the number of proteins asso ciated with E. coli 55 RNA. In addition to L5, LIB, L25, L2 and L17, proteins L15, L16, L22, L33, L3A and variable amounts of L3 and L21 were bound (Metspalu et al, 1982a). These results, as well as the results of our experiments, clearly indicate that the binding of E. coli 505 ribosomal proteins to immobilized E. coli 55 RNA depends on the ionic conditions and protein concentration used.…”

“…This gave a spacer arm of about 30A, which is three times longer than the spacer used in our experiments. The binding of E. coli 50S proteins to E. coli 55 RNA immobilized by this method was studied using different ionic conditions and protein con centrations (Metspalu et al, 1982a). At a ribosomal protein con centration of 0.2mg/mL, and with binding buffer containing 30 mM MgCl2 and 200 mM KCl, proteins L5, L18, L25 and small amounts of L2 bound to the E. coli 55 RNA.…”

“…From the results of these affinity chromatography experiments it was not possible to determine if bound proteins are interacting directly with RNA or binding indirectly by association with proteins L5, L18 and L25 which have been shown to interact directly with the RNA (Spierer and Zimmermann, 1978b). Metspalu et al (1982a) Figure 7). However, at lower salt concentrations C0.2M KCl) proteins L2, L17 and variable amounts of L16 were bound to both yeast 55 RNA and yeast 5.8 RNA (Figures 8, 9, 10j.…”

500‐MHz 1H‐NMR Studies of Ribosomal Proteins Isolated from 70‐S Ribosomes of Escherichia coli

“…It has been shown however, that such procedures do not lead to irreversible denaturation. The denatured material isolated in this way can still be used succesfully in, for example, reconstitution experiments [12] and affinity chromatography [13]. For NMR studies, where the aim is to obtain information about the three-dimensional structure of proteins, it is essential to use a procedure which yields material that has remained native or that can be renatured.…”