Role of the 30S ribosomal subunit, initiation factors, and specific ion concentration in barotolerant protein synthesis in Pseudomonas bathycetes

Abstract

Washed (1 M NH4Cl) ribosomes from Pseudomonas bathycetes, Pseudomonas fluorescens, and Escherichia coli were tested for their ability to synthesize protein or polypeptide at high pressure when used as such, when recombined with homologous initiation factors, and when recombined with heterologous initiation factors. The responses of natural messenger ribonucleic acid (MS-2)-directed systems to pressure were independent of the source of initiation factors and paralleled those of the washed ribosomes in polyuridylate-directed systems. In all cases, the responses to pressure were parallel to those obtained when unwashed ribosomes were utilized; therefore, we concluded that the initiation factors were interchangeable among these organisms, and that these factors did not play a critical role in determining the pressure responses of the protein-synthesizing systems. P. bathycetes ribosomal subunits were isolated under a variety of ionic conditions. These were tested for their ability to synthesize protein and polyphenylalanine at a variety of pressures when used in reconstituted P. bathycetes homologous systems and in hybrid systems with ribosomal subunits from E. coli and P. fluorescens. O. bathycetes 30S subunits, isolated in a buffer solution containing 0 mM NaCl and O mM KC] were functional at any pressure; those isolated in the presence of 150 mM NaCl and 0 mM KCl were functional at 1 atmosphere but barosensitive, and those isolated in the presence of O mM NaCl and 150 mM KCl retained the ion-mediated barotolerance characteristic of crude P. bathycetes ribosome preparations. The 50S subunit remained functional regardless of the method of isolation, and it had no effect on pressure sensitivity.