Geometry of the Protein S4 from Escherichia coli Ribosomes

Abstract

The shape of protein S4 from Escherichia coli ribosomes in solution was determined by hydrodynamic methods and low-angle X-ray scattering. The molecular weight of 24000 determined by low-angle X-ray scattering is within 3 % of that found by sedimentation equilibrium analysis and 8 of that determined by amino acid sequence work. The radius of gyration of 3.36 nm. the radius of gyration of the cross section of 0.41 nm and the hydrodynamic studies revealed that protein S4 is not spherical, but rather has a markedly extended shape. Calculations of different conformations, e.g. random coil, based on the parameters evaluated from hydrodynamic methods, revealed a rod-like structure of S4 with a length of 14 nm and a diameter of 1 nm. This is supported by a model of an equivalent scattering particle of uniform density based on all parameters obtained in this study.Physiochemical characterizations of ribosomal proteins, especially those which specifically bind to 23-S, 16-S or 5-S RNA, are of importance for studies on protein-RNA interactions and on the topography of the ribosoinal particle. Although the chemical and immunological properties of the isolated proteins [l -51 have been studied intensively, very little is known about the shape of the individual proteins, e.g. if they are globular or highly asymmetric. Only the shapes of the proteins L7 and L12 have recently been determined [6].Protein S4, of which the primary sequence has been determined [7], is one of the most interesting proteins in the Escherichiu coli 30-S ribosomal subunits. It binds specifically to the 16-S RNA [8], and details of the interaction between proteins S4 and 16-S RNA have been studied [9-131. Furthermore, mutants with drastically altered S4 proteins, whose binding to 16-S RNA can be impaired, have been investigated [14-211. Information on the shape of protein S4 would help to understand its role in protein-RNA interactions.In the present study attempts were made to determine the shape of protein S4 in solution, e . g . reconstitution buffer [22]. The gross conformation of the S4 molecule in solution was determined by different techniques, of which low-angle X-ray scattering proved to be the most useful. Moreover, several hydrodynamic methods revealed that S4 has a markedly extended shape. MATERIALS AND METHODS Isolation of Protein S4

On the Renaturation of Ribosomal Protein L11

“…As an example one might cite the values found for the radius of gyration of protein S4 in non-denaturing buffers: 1.8 nni [I], 2.6 nm [2], 3.35 nm [3] and 4.2 nm [4]. It is not reasonable to attribute variations of this magnitude to technical difficulties.…”

Quaternary structure of the ribosomal 30S subunit: model and its experimental testing.

“…First, no information was available as to three-dimensional structure and morphology of the 16S RNA; this RNA is known to be a structural backbone for arrangement of ribosomal proteins. Second, immunoelectron microscopy (9, 10) and neutron scattering (14,15) data on proteins within the 30S subunit, as well as some physical measurements of isolated ribosomal proteins (16)(17)(18)(19)(20)(21), have suggested strongly elongated or very expanded shapes of many ribosomal proteins; this led to ambiguity in interpretation of the results on chemical crosslinking, energy transfer, etc.…”

A Trypsin‐Resistant Fragment from Complexes of Ribosomal Protein S4 with 16‐S RNA of Escherichia coli and from the Uncomplexed Protein

“…Hydrodynamic studies [4], low-angle X-ray scattering studies [5,6] and the electron microscopic visualisation of ribosome-bound antibody markers [7,8] all indicate that the protein has a highly asymmetric structure. Furthermore, nuclear magnetic resonance and other spectroscopic studies [9,10] have indicated that the protein has an open and extended structure with about 30 % a-helix and little p-structure.…”