Unusual rRNA-linked complex of 50S ribosomal subunits isolated from an Escherichia coli RNase III mutant.

We have isolated and characterized complexes of ribosomal subunits from Escherichia coli mutant AB301-105 connected by strands of unprocessed RNA. By electron microscopy of these complexes, the location of the 5' end of 5S RNA was established and the location of the 3' end of 23S RNA was confirmed. We also note that in these complexes insertion of 5S rRNA can proceed without the 23S-5S spacer having been processed.     

Properties of ribosomes from erythromycin resistant mutants of Escherichia coli.

Summary We have studied the in vitro properties of ribosomes from several mutants resistant to erythromycin. Mutations in three different genes may confer resistance to erythromycin. Two of them are structural genes for proteins L4 and L22 of the large subunit. The third mutation (in eryC gene) seems to affect mainly the small subunit. The mechanism of action of the antibiotic may involve both subunits.     

Structural and functional studies of ribonucleoprotein fragments isolated from Escherichia coli 50 S ribosomal subunits.

Ribonuclease digestion of 50 S-derived LiCl cores led to 22 ribonucleoprotein particles which were isolated by repeated sucrose gradient centrifugations. The protein content was determined and ranged from 2 to 28 proteins. Most of the fragments showed a unique RNA pattern as judged by acrylamide gel electrophoresis.Functional tests were performed with selected fragments. No fragment was active in the poly(U) or the peptidyl-transferase assay. Chloramphenicol binding studies revealed that in addition to the dominant role of protein L16, the protein L11 (or L6) is involved directly in the drug binding. Finally, tests for ATPase and GTPase activity showed that protein L18 is involved in GTPase activity.     

Biochemical and genetic studies on two different types of erythromycin resistant mutants of Escherichia coli with altered ribosomal proteins

Summary Ribosomes from nine E. coli mutants with high level resistance to the antibiotic erythromycin were isolated and their proteins were compared with those of the parental strains by two-dimensional polyacrylamide gel electrophoresis, by carboxymethylcellulose column chromatography and by immunological techniques. Two 50S proteins were found to be altered in the mutants: either L 4 or L 22.Ribosomes with an altered L4 protein bound erythromycin rather poorly and the formation of N-acetylphenylalanyl puromycin was drastically decreased. On the other handribosomes with an altered L22 protein bound erythromycin as efficiently as wild type ribosomes and their puromycin reaction was at least as good as that of wild type ribosomes.Transduction experiments showed that the mutations affecting both proteins, L4 and L22, are located very close to the str and spc genes, nearer to the spc than to str gene.Paper No. 61 on Ribosomal Proteins. Preceding paper is by Hasenbank et al., Molec. gen. Genet., 127, 1–18 (1973).Communicated by E. Bautz     

Characterization of ribonucleoprotein subparticles from 50 S ribosomal subunits of Escherichia coli.

Large ribonucleoprotein subparticles were recovered upon ribonuclease digestion of the 50 S ribosomal subunits of Escherichia coli, partially deproteinized by LiCl. Both their RNA and their protein compositions were analysed. The subunits, treated with LiCl at a concentration of 5.5 m, released an homogeneous subparticle containing proteins L₃, L₄, L₁₃, L₁₇, L₂₂ and L₂₉, about 70% of the 13 S fragment of 23 S RNA and about 50% of the 18 S one. Slightly larger species of subparticles were obtained from 50 S subunits treated with LiCl at concentrations between 3 m and 5 m; they contained in addition proteins L₂₀, L₂₁ and L₂₃ or L₂, L₁₄, L₂₀, L₂₁ and L₂₃ and a few small 23 S RNA fragments. No large subparticle was recovered from the 6 m-LiCl-treated 50 S subunits which contain only proteins L₃, L₁₃ and L₁₇. These LiCl subparticles were compared with those obtained from intact, unfolded and sodium doecyl sulphatetreated 50 S subunits.These studies reveal that in the presence of 0.10 m-magnesium acetate there is a very compact area within 50 S subunits consisting of proteins L₃, L₄, L₁₃, L₁₇, L₂₂ and L₂₉ and of about 60% of 23 S RNA; this area probably has an essential structural role. The results also show that 23 S RNA has a more folded conformation when within the 50 S subunit than when isolated, this conformation being stabilized by some of the 50 S proteins, in particular proteins L₄, L₂₂, L₂₀ and L₂₁. Finally these data permit a more definite localization of the primary and/or secondary binding sites of proteins L₂, L₃, L₄, L₁₄, L₁₇, L₂₀, L₂₁ and L₂₂ on 23 S RNA.     

The use of sym-triazine trichloride in RNA-protein cross-linking studies with Escherichia coli ribosomal subunits.

Summary The reagent sym-triazine trichloride is used as a bifunctional reagent to generate RNA-protein cross-links within intact ribosomal subunits from E. coli. The reaction takes place in a stepwise manner, involving substitution of one chlorine atom at 12° and pH 8, and substitution of the second at 40° and pH 6. The cross-linked proteins are analysed by two-dimensional electrophoresis, and the existence of a stable cross-linkage is demonstrated by isolating protein-oligonucleotide complexes from ³²P-labelled subunits. The proteins cross-linked are S3 and S4 in the 30S subunit, and L2 in the large subunit, together with smaller amounts of other proteins. The reagent should prove useful in topographical studies of the E. coli ribosome as it is a rigid molecule and generates very short cross-links.     

Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions.     

Negatively stained 50 S ribosomal subunits of Escherichia coli

Ribosomes are large nucleoproteins of approximately 3 X 10(6) Mr. In contrast to helical or spherical nucleoproteins (viruses) of similar size (which consist of several hundred small asymmetric units reproduced by symmetry), ribosomes are completely asymmetric; therefore, the amount of structural information (defined by the number of independent image elements) necessarily increases from about 10 to 20 to about 1000 to 2000 (at resolutions of the order of 2 nm). With present techniques, only stained single particles can be studied in the electron microscope. Our published work on the 30 S subunit and on the 50 S subunit has demonstrated that three-dimensional reconstructions of stained single particles show a great number of structural details that are reproducible if the particles have the same orientation. One of the main results of this paper is the final proof of this reproducibility from detailed comparisons of individual 50 S subunits and of independent averages over a few (3 to 5) particles in the kidney or crown orientation; in the latter case, even after a chemical modification. The 50 S subunit is non-uniformly stained along the optical axis. It displays a complicated, stain-filled channel-like structure, within which is approximately the partial volume expected for the RNA. The particle shows an irregular but reproducible boundary surface against the stain. At several sites, the channel structure protrudes to the surface. Since the secondary structure of the RNA is well known, one might try to locate it in the subunit after chemical identification of its surface contacts (the 3' end of 23 S RNA and the 3' end of the 5 S RNA have been localized). Most interesting is a groove on the surface, which might accommodate the mRNA.     

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.     

A new ribosomal mutation which affects the two ribosomal subunits in Escherichia coli.

Summary The nif cistrons indentified by complementation analysis in the preceding paper (Dixon et al., 1977) were mapped with respect to hisD and to each other by Pl cotransduction and three-factor reciprocal crosses. The order obtained was hisD nifB nifA (nifL) nifF nifE nifK nifD nifH. Analysis of hisD2-nif cotransduction data by the Wu equation (Wu, 1966) suggested that the nif genes are divided into two clusters: a his-proximal cluster comprising nifBA(L)F and a his-distal group of nifEKDH.     

Characterization of a mutant form of ribosomal protein S1 from Escherichia coli.

An altered form of ribosomal protein S1 from a mutant of Escherichia coli has been isolated and characterized. The mutant protein (denoted m1-S1) has a molecular weight of 57,000 as shown by sodium dodecyl sulfate-gel electrophoresis and the same NH2-terminal sequence as wild type S1. Protein m1-S1 binds poly(U) in the same manner as protein S1 and is active in protein synthesis with either synthetic or natural mRNA. Thus, about 75% of the sequence of protein S1 (which includes the NH2-terminal region) contains essentially all the functional domains of this protein involved in protein biosynthesis.