Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.

Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature.

Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins of Lactococci, Streptococci, and Pediococci.

Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins are equally effective procedures for detecting differences and similarities in the Lactococci, Streptococci and Pediococci examined. Electrophoretic distribution patterns of ribosomal subunit proteins may be a useful tool in taxonomic studies.

Methylated amino acids in the proteins of the cytoplasmic ribosome of Tetrahymena thermophila.

Two-dimensional electrophoretic analysis of proteins of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila labeled in vivo with L-[14C1]methionine and L-[3H-methyl]methionine identifies one heavily methylated protein in each ribosomal subunit. These proteins, S31 and L21, each contain N epsilon-trimethyl-lysine.

Ribosomal proteins of Tetrahymena thermophila. Correlation of one- and two-dimensional electrophoretic migration patterns and characterization of additional small and large subunit proteins.

Further analysis of the protein complement of the cytoplasmic ribosome of the protozoon Tetrahymena thermophila has led to the identification and characterization of seven additional proteins, three in the small and four in the large subunit of this ribosome. Several of these proteins are poorly soluble or insoluble in the absence of high concentrations of urea and are not seen in the electrophoretic distribution patterns of ribosomal proteins in two-dimensional polyacrylamide gels unless 6 M urea is added to electrode buffers in contact with protein samples (first dimension) and first-dimension gels (second dimension). The migration patterns of the 40S and 60S subunits of the T. thermophila ribosome in one-dimensional polyacrylamide SDS gels and in two-dimensional gels prepared by means of the basic-acidic system of Kaltschmidt and Wittmann**, and the basic-SDS system of Zinker and Warner*** have been correlated.

Protein-RNA crosslinking in the subunits of the cytoplasmic ribosome of Tetrahymena thermophila.

Use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to introduce RNA-protein crosslinks in the 40S and 60S subunits of the cytoplasmic ribosome of Tetrahymena thermophila is described, and proteins linked covalently to 17S and 26S ribosomal RNAs are identified. RNA-protein crosslinking is accompanied by extensive dimerization and aggregation of ribosomal subunits probably due to formation of interparticle protein-protein crosslinks.

Comparison of active and inactive forms of the E. coli 30S ribosomal subunits.

Dissociation of E. Coli 70S ribosomes in the presence of 0.1 mM Mg++ yields partially inactivated 30S and 50S subunits. This inactivation can be avoided by dissociating the 70S ribosome in a medium containing 10 mM Mg++. 400 mM Na+. Comparison of the active and inactive forms of the 30S and 50S subunits has led to the following conclusions: 1) The two forms possess identical (50S subunits) or very similar (30S subunits) hydrodynamic properties. No differences in their morphologies is detectable by electron microscopy. 2) They possess the same protein compositions except for the presence of a larger amount of protein S1 in the inactive than in the active form of the 30S subunit. 3) They differ significantly in functional properties: more efficient association of the active than of the inactive forms with the complementary subunit; extensive dimerization of inactive 30S subunits in the presence of 10 mM Mg++; no dimerization of active 30S subunits under the same conditions; six-fold higher peptidyl transferase activity of active as compared to inactive 50S subunits.

5S RNA-protein complexes released by EDTA treatment of 60S ribosomal subunits of Tetrahymena thermophila.

Treatment of large (60S) subunit of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila with EDTA causes quantitative release of 5S rRNA associated with variable non quantitative amounts of one or more of 60S proteins L4, L15, L24, L31 and L41. The composition of the group of proteins released with 5S rRNA depends on both the molar ratio of EDTA and 60S subunits and the concentration of 60S subunits, in treatment mixtures.

Ribosomal subunits and ribosomal proteins of Tetrahymena thermophila. Effect of the presence of iodoacetamide during ribosome extraction on the properties of the subunits.

Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymena thermophila and the isolated subunits are inactive. Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits. The protein complements of these subunits have been characterized in two different two-dimensional electrophoretic systems, and their molecular weights have been determined.

Concerning the thermal stability of E. coli 23S ribosomal RNA.

Total RNA prepared from E. coli by several extraction procedures behaves as a mixture of covalently continuous heat stable 23S, 16S and 4-5S components. 16S rRNA remains heat stable after isolation from such preparations, whereas isolated 23S rRNA is heat labile but becomes heat stable after EDTA treatment. This and other evidence leads to the conclusion that heat lability of purified 23S rRNA is due, not to nuclease contamination of the type observed in earlier studies of the stability of this RNA, but to polyvalent cation catalyzed temperature-dependent scission of phosphodiester bonds. Heat stability of 23S rRNA in total RNA is due to the presence in these preparations of a contaminant which appears to act as a chelator of polyvalent cations. This material is similar or identical to the pyrogenic E. coli lipopolysaccharide described by Westphal and coll.

Partial deproteinization of ribosomal subunits by treatment with high concentrations of sodium chloride: physical and chemical characterization of protein depleted particles.

Sedimentation of E. coli ribosomal subunits through sucrose gradients containing high concentrations of NaCl (0.1 - 1.0 M) brings about removal of a specific fraction of their proteins. The alterations in the structural properties of the subunits caused by dissociation of these proteins are studied. They appear to be, at least partly the result of removal of constraints on RNA conformation imposed by the released proteins.

Effects of partial deproteinization on the functional properties of 50S ribosomal subunits of E. coli.

Sedimentation of E. coli 50S ribosomal subunits through sucrose gradients containing 10 mM Mg2+ and high concentrations of NaCl (0.1-1.0 M) leads to removal of proteins L16 and L25. Analyses of the structural and functional properties of the protein depleted particles shows that removal of L16 and L25 from the 50S subunit causes loss of its ability to bind tRNA, to associate with the 30S subunit and to catalyze peptide bond formation. Reassociation of both L16 and L25 with core particles lacking these proteins is necessary for recovery of peptidyl transferase activity.

Reconstitution of active 30-S ribosomal subunits in vitro using heat-denatured 16-S rRNA.

30-S ribosomal subunits which have been reconstituted using heat-denatured 16-S rRNA can participate in the synthesis of lysosyme in vitro. Therefore all the information contributed by 16-S rRNA to the reconstitution process is carried in the primary sequence of this RNA. The specific protein-synthesizing activity of 30-S subunits reconstituted from 30-S subunit proteins and heat-denatured 16-S rRNA is about one third of that observed if unheated 16-S rRNA is used and is comparable to the activity of 30-S particles isolated after dissociation of 70-S ribosomes in the presence of 0.1 mM Mg2+.

The effect of high ionic strength on the sedimentation properties of the biologically active component of bacterial ribonucleic acid.

1. The sedimentation properties of the fraction of bacterial RNA which stimulates the incorporation of amino acids into acid-insoluble material in vitro depend on the ionic strength of the sedimentation medium. 2. The different distributions of stimulatory activity found in centrifuged linear sucrose gradients loaded with bacterial RNA and containing 0.1m- or 0.6m-sodium chloride resemble closely the different sedimentation profiles of rapidly labelled RNA observed under the same conditions. 3. These findings agree with those of previous work of a similar nature (Willson & Gros, 1964) and demonstrate that the component of bacterial RNA preparations which stimulates the incorporation of amino acids into acid-insoluble peptides in vitro is contained in their rapidly labelled fraction.