Structure and function of bacterial ribosomes: XII. Accumulation of 21 s particles by some cold-sensitive mutants of Escherichia coli

Abstract

A spontaneous spectinomycin resistant mutant strain Spc-49-1 which has been derived from Escherichia coli strain NO49 is unable to grow at 20 °C. The mutant is also defective in ribosome assembly at 20 °C. In Tryptone broth, at 20 °C, it is unable to synthesize either 30 s or 50 s subunits and accumulates two kinds of incomplete particles, 21s and “30 s” particles, which are related to 30 s and 50 s subunits, respectively. These particles have been shown to be converted to 30 s and 50 s subunits, respectively, after subsequent incubation at 42 °C. It appears that both 21 s and “30 s” particles are normal intermediates in biosynthesis of ribosomes or related to such intermediates. The 21 s particles labeled with radioactive amino acids were isolated and their protein composition was analyzed. The following proteins were found: P3b,c, P4a, P4b, P5, P9, P10a, P10b, P13 and P14. P8 and P12 were found in some preparations. Other proteins (P1, P2, P3, P4, P6, P7, P10, P11 and P15) were not present in significant amounts. All the proteins found in the 21 s particles are in the early part of the in vitro assembly map constructed previously using in vitro 30 s ribosomal reconstitution. It is concluded that the order of addition of proteins during in vivo 30 s assembly is similar or identical to the order of addition of proteins during in vitro reconstitution of 30 s subunits. The mutation in the strain Sp-49-1 responsible for the cold-sensitive and ribosome assembly defective phenotypes was characterized. Like other spc r loci, the altered locus, spc-49-1, appears to map between aroE and str loci. The altered protein component was identified as the 30 s ribosomal protein P4, the protein studied previously in spectinomycin-resistant mutant. Possible mechanisms by which mutations affect ribosome assembly are discussed.