Akira Muto

The number of ribosomal RNA genes in Mycoplasma capricolum

SummaryWe have examined the number of rRNA genes in Mycoplasma capricolum (KID) by hybridization of BglII-, EcoRI- and XbaI-digests of DNA to [3′-32P] 16S, 23S and 5S rRNAs according to the Southern procedure (1975). All the restriction gels gave two radioactive bands with three kinds of rRNA. Furthermore, band positions were indistinguishable from one another when 16S, 23S and 5S rRNAs were used as probes, indicating that each band contains sequences corresponding to the 3′-termini of 16S, 23S and 5S rRNAs. It is thus concluded that Mycoplasma capricolum chromosome carries at least two sets of genes for 16S, 23S and 5S rRNAs.

Messenger activity of nascent ribosomal RNA

Abstract Escherichia coli nascent rRNA containing little contamination of messenger RNA and mature RNA was prepared from chloramphenicol-particles or relaxed-particles. Cell-free amino acid-incorporation experiments using the purified nascent rRNA preparations showed that the messenger activities of 17 and 23 s nascent rRNA were six and three times those of 16 and 23 s mature rRNA, respectively. Protein synthesis directed by the nascent rRNA took place on 70 s ribosomes with which a part of the added nascent rRNA combined. Analyses by polyacrylamide gel electrophoresis of the products formed during cell-free protein synthesis in the presence of nascent rRNA showed that the bulk of those products had mobilities similar to those of ribosomal proteins and distinct to those of the products of messenger RNA-directed synthesis. It was concluded from these facts that nascent rRNA acts as a template for some polypeptide synthesis in vitro.

Effects of some antibiotics on the stringent control of RNA synthesis in Escherichia coli.

SummaryEffects of neomycin, spectinomycin, tetracycline and chloramphenicol on the stringent control RNA synthesis and on ppGpp synthesis in the rel+-cells of Escherichia coli having a temperature-sensitive valyl-tRNA synthetase were examined. Without antibiotics, ppGpp began to accumulate and both RNA and protein syntheses were inhibited by transferring the exponentially growing cells from 30°C (permissive temp.) to 40°C (non-permissive temp.). Tetracycline or chloramphenicol, when added after the temperature shift, caused a resumtion of RNA synthesis and decay of the accumulated ppGpp, while neomycin or spectinomycin had little effect both on RNA synthesis and the level of ppGpp. When the cells were treated with these antibiotics at permissive temperature, the shift of the temperature to 40°C caused neither inhibition of RNA synthesis nor an accumulation of ppGpp. When neomycin or spectinomycin was added at the beginning of the temperature shift, RNA synthesis continued with an accumulation of ppGpp. Tetracycline or chloramphenicol had no such effect under the same conditions; RNA synthesis continued without an accumulation of ppGpp.

Protein synthesis in a relaxed-control mutant of Escherichia coli upon recovery from methionine starvation

The site of protein synthesis in cells of a methionine-requiring RCre1 -mutant of Escherichia coli during recovery from methionine starvation has been investigated in vivo and in vitro. The cells accumulate a considerable amount of “RC-particles” during the course of methionine starvation. Upon restoration of methionine, the cells synthesize preferentially ribosomal protein. A pulse-label of [3H]leucine in such cells recovering from methionine starvation resulted in a preferential labelling of nascent protein combined with 70 s ribosomes. The protein was then transferred to the soluble fraction and RC-particles. More than a half of this protein is ultimately built up to ribosomal particles. Free RC-particles are not the primary site of protein synthesis. The sites of incorporation of [14C]Iysine into protein in an extract prepared from cells recovering from methionine starvation were found to be exclusively 70 s ribosomes and “polysomes”. Free RC-particles do not incorporate [14C]lysine in this system. A part of the RC-particle has been found combined in cell extracts with 70 s ribosomes, forming “heavy 70 s” and polysome-like material.

Preferential ribosomal RNA synthesis in the lysate of Escherichia coli.

SummaryThe RNA synthesis in non-viscous lysates containing the intact folded chromosome and cytoplasm fractions prepared from Escherichia coli has been examined in vitro. The RNA synthesis not only by chain extension but also by new chain initiation occurs in this system. While the RNA synthesis by chain extension takes place on the chromosome fraction alone (Pettijohn et al. 1970), an addition of the cytoplasm fraction is necessary for the synthesis by new chain initiations (de novo synthesis).Analyses of the in vitro synthesized RNA by hybridization-competition and by sucrose gradient centrifugation show that 16S and 23S ribosomal RNAs account for about 40% of the total RNA products. The cytoplasm fraction is required for the de novo synthesis of ribosomal RNA at high relative rate. Guanosine tetraphosphate (ppGpp) does not specifically inhibit ribosomal RNA synthesis in this system.

Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis

SummaryA temperature-sensitive EFG mutant of Bacillus subtilis was isolated and characterized. This mutant, ts32, synthesizes stable RNA at 48° C with or at 50° C without accompanied protein synthesis. The initial rate of the RNA synthesis at 48° C or 50° C was 1.5 to 2.0 times as much as that at 30° C.This mutant as well as its parent (both leu-) showed stringent response for the RNA synthesis upon deprivation of amino acids with an accumulation of the MS nucleotides (pp Gpp and pppGpp). On raising temperature to 48° C or 50° C, the ts-cells immediately began to synthesize the stable RNA with an initial increase of the MS nucleotides. No drastic decrease in amount of the MS was observed during the active RNA synthesis.These results suggest that EFG is somehow involved in repressing the stable RNA synthesis, and have broken the close relationship between the stable RNA synthesis and the MS nucleotides hitherto reported.

Control of ribosomal RNA synthesis in Escherichia coli

SummaryThe ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the “4S” fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation of rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acidstarved rel+ cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.

Correlation of 30S ribosomal proteins of Escherichia coli fractionated on carboxymethyl-cellulose column chromatography to the standard nomenclature.

SummaryThe nomenclature proposed by Otaka et al. (1968) for the 30S ribosomal protein components of Escherichia coli as separated by carboxymethyl(CM)-cellulose column chromatography was adopted in several papers in which the genetic loci for many 30S ribosomal proteins on the E. coli chromosome were determined. In order to compare these data with those obtained in other laboratories, the 30S ribosomal proteins fractionated by CM-cellulose chromatography were correlated with the standard nomenclature proposed by Wittmann et al. (1971).

Chemical and genetic analysis of 16S ribosomal RNA in Escherichia coli

SummaryComparative chemical analyses of oligonucleotides arising from pancreatic RNase digestions of 16S ribosomal RNAs from Escherichia coli strains K12 and B(H) showed that a decanucleotide fragment, (5Ap,4Gp)Cp, could be detected exclusively in strain K12 but not in strain B(H), in spite of gross similarity of nucleotide distributions between the two strains.The K12-specific oligonucleotide could not be cotransduced with streptomycin and/or spectinomycin resistant markers from K12 to B(H) by phage Plkc, indicating that the genes specifying 16S ribosomal RNA are not closely linked to these markers on the chromosome.

Ribosomal RNA synthesis in the F′14 episome-containing minicells of Escherichia coli

SummaryF′14 episome of E. coli, which includes genes for rRNAs but not the “ribosomal protein operon” (Nomura and Engbeak, 1972) has been transferred to a minicell-producing strain of E. coli K12. The RNA formed in the purified episome-containing minicells was identified as rRNAs by sedimentation analyses and DNA-RNA hybridization-competition experiments. It is concluded that the rRNA genes in the F′14 episome can be transcribed without expression of the “ribosomal protein operon”.