Quaternary structure of the small ribosomal subunit ofEscherichia colidetermined by neutron diffraction

Abstract

A complete three-dimensional mapping of the 21 proteins of the small ribosomal subunit of E. coli has been generated from 105 interprotein distance measurements derived from small-angle neutron scattering data, obtained in solution. A statistical analysis of the map and comparison with the results of similar mappings, using other biochemical or biophysical measurements, are presented.In all living organisms, the process of protein synthesis is mediated by a complex ribonucleoprotein organelle, the ribosome. The ribosome is comprised of a large and a small subunit. The large ribosomal subunit of the bacterium Escherichia coli consists of 32 different proteins and two ribonucleic acid (rRNA) molecules. The small ribosomal (30S) subunit has 21 unique polypeptides and a single rRNA (Giri, Hill & Wittmann, 1984;Wittmann-Leibold, 1986). Approximately one-third of the total mass of the ribosome is protein and the rest RNA. While much is known of the function of the ribosome, from a phenomenological viewpoint we lack a detailed picture of its threedimensional structure, and as a result detailed mechanistic interpretations of ribosome function have not been developed.The quaternary organizations of the ribosomal proteins and RNA's have been investigated by many techniques, including immune-electron microscopy (Lake, 1985; Stoeffier & Stoeffler-Meilicke, 1986), fluorescence energy transfer (Huang, Fairclough & Cantor, 1975), bifunctional chemical crosslinking (Brimacombe, Maly & Zweib, 1983). We have constructed a map of the relative 3-space positions of the proteins of the small ribosomal subunit from a large set of interprotein distances obtained from neutron diffraction measurements of 30S subunits in solution (Moore, Langer, Schoenborn & Engelman, 1977;Langer, Engelman & Moore, 1978;Schindler, Langer, Engelman & Moore, 1979;Ramakrishnan, Capel, Kjeldgaard, Engelman & Moore, 1984;Capel, et al., 1987;Capel, Kjeldgaard, Engelman & Moore, 1988).The method used to abstract interprotein distance information from solution scattering data Moore, 1979;Ramakrishnan & Moore, 1981) relies upon the following facts: (1) ribosomes can be disaggregated into their constituent proteins and rRNAs, which can be purified in high yield; (2) it is possible to reconstitute intact active ribosomal particles from purified proteins and rRNAs; and (3) prokaryotes can subsist on media containing deuterium instead of hydrogen.These facts enable us to generate reconstituted 30S ribosomal particles in which specific pairs of proteins are labelled with the hydrogen isotope opposite to that present in the bulk particle. By measuring the neutron scattering intensities of isotopically labelled 30S ribosomal subunits in solution that is contrast matched to their bulk scattering-length density one can obtain a good estimate of the partial scattering function, whose properties are modulated by the distance between and the shapes of the labelled pair of proteins. This partial scattering function is a damped sinusoid in form and is usually referred t...