Katsumi Isono

An improved method for two-dimensional gel-electrophoresis: analysis of mutationally altered ribosomal proteins of Escherichia coli.

SummaryAn improved method for the two-dimensional electrophoretic analysis of ribosomal proteins on acrylamide gel slabs has been developed by combining the procedures for the first dimension of Mets and Bogorad (1974) and for the second dimension of Kaltschmidt and Wittmann (1970) and by introducing several modification. Ribosomal proteins of various Escherichia coli mutants have been analyzed by the new method. Advantages are that (1) it requires only small amounts of protein (100–200 μg 70S ribosomal proteins), (2) reproducibility is very high, and (3) it makes it easier to identify mutational alterations in proteins S10, L4, L10, and L21 which hardly migrate out of the sample gel with our previous electrophoresis procedure. Furthermore, the new method can be nicely adapted to analysis of the ribosomal proteins from other organisms, such as Bacilli or yeast.

Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

SummaryThe ribosomal proteins of temperature-sensitive mutants of Escherichia coli isolated independently after mutagenesis with nitrosoguanidine were analyzed by two-dimensional gel electrophoresis. Out of 400 mutants analyzed, 60 mutants (15%) showed alterations in a total of 22 different ribosomal proteins. The proteins altered in these mutants are S2, S4, S6, S7, S8, S10, S15, S16, S18, L1, L3, L6, L10, L11, L14, L15, L17, L18, L19, L22, L23 and L24. A large number of them (25 mutants) have mutations in protein S4 of the small subunit, while four mutants showed alterations in protein L6 of the large subunit. The importance of these mutants for structural and functional analyses of ribosomes is discussed.

Ribosomal protein modification in Escherichia coli: I. A mutant lacking the N-terminal acetylation of protein S5 exhibits thermosensitivity

Abstract A thermosensitive mutant (JE386) of Escherichia coli which harbours an alteration in protein S5 of the smaller ribosomal subunit has been isolated. Genetic studies have shown that the lesion causing thermosensitivity also causes the alteration in protein S5, and that this mutation is not in the structural gene for S5 ( rpsE ). Hence the mutation has been termed rimJ (ribosomal modification). Protein-chemical studies of protein S5 purified from JE386 and its wild-type parent indicated an alteration in the N-terminal tryptic peptide. Amino acid sequence analysis of the N-terminal peptides showed complete homology between wild-type and mutant, suggesting that the N-terminal modification (acetylation) of the parent was absent in the mutant. Gradient transmission mapping has located the rimJ mutation at 31 minutes on the current E. coli genetic map. By constructing a derivative of the mutant heterozygous for rimJ , it has been found that the wild-type allele is dominant over the mutant one. Ts + revertants of JE386 have been isolated which show either a wild-type ribosomal protein electrophoresis pattern, or an additional alteration in either protein S4 or S5. The mutations in S4 and S5 may compensate the lesion caused by the rimJ mutation of JE386, that is even though the N-terminus of S5 remains unacetylated, bacteria can grow at 42 °C. Furthermore, a mutation near or at strA carried by JE386 has been found to be involved in the phenotypic expression of the rimJ mutation. This mutation was also found to be present in four other strA mutants. Possible implications of the modification of ribosomal proteins in vivo are discussed.

Further temperature-sensitive mutants ofEscherichia coli with altered ribosomal proteins

SummaryVarious alterations in ribosomal proteins were detected in forty-one mutants ofE. coli isolated as temperature-sensitive mutants. Out of these, six are new classes of mutants harboring mutations in proteins S3, L5, L7 (L12), L29, L30 and L33. One of them apparently lacks protein L7 of the large subunit. These mutants together with those reported previously (Isono et al., 1976) total one hundred and one ribosomal mutants in thirty different proteins.

Mutations affecting the structural genes and the genes coding for modifying enzymes for ribosomal proteins in Escherichia coli

SummaryTemperature-sensitive mutants of an Escherichia coli K-12 strain PA3092 have been isolated following mutagenesis with nitrosoguanidine, and their ribosomal proteins analyzed by two-dimensional gel electrophoresis This method was found to be very efficient in obtaining mutants with various structural alterations in ribosomal proteins. Thus a total of some 160 mutants with alterations in 41 different ribosomal proteins have so far been isolated. By characterizing these mutants, we could isolate, not only those mutants with alterations in the structural genes for various ribosomal proteins, but also those with impairments in the modification of proteins S5, S18 and L12. Furthermore, a mutant has been obtained which apparently lacks the protein S20 (L26) with a concomitant reduction to a great extent of the polypeptide synthetic activity of the small subunit. The usefulness of these mutants in establishing the genetic architecture of the genes coding for the ribosomal proteins and their modifiers is discussed.

Cloning and the nucleotide sequence of the genes for Escherichia coli ribosomal proteins L28 (rpmB) and L33 (rpmG).

SummaryThe specialized transducing bacteriophage λdpyrE DNA was used as a source of DNA to clone two ribosomal protein genes rpmB (L28) and rpmG (L33) on the cloning vehicle pACYC184. Using one of these plasmids, the nucleotide sequence of these two genes and their flanking regions were determined. The amino acid sequences of both proteins deduced from the nucleotide sequences match with the amino acid sequences previously determined, with one exception. The nucleotide sequences suggest that these two ribosomal protein genes are cotranstribed. There was no expression of the second gene of the operon, rpmG, in the absence of the 5′ sequences adjacent to the first gene, rmpB. Observation of the structure of mRNA also strongly supports the idea that rpmB and rpmG are in a single transcription unit whose order is: rpmBp-rpmB-rpmG-rpmGt.

Ribosomal protein modification in Escherichia coli

SummaryAmong mutants of E. coli selected for temperaturesensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the acetylase activity specific for protein L12 was negligible, when assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL that codes for an acetylating enzyme specific for ribosomal protein L12.

An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli

SummaryAn amber mutation has been induced in the gene rpsA (which codes fo ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.

Correlation between 30S ribosomal proteins of Bacillus stearothermophilus and Escherichia coli.

SummaryThe 30S ribosomal proteins from Bacillus stearothermophilus strains 799 and 10 were purified and correlated with those from E. coli by comparing their two-dimensional electrophoretic mobility, immunological cross-reaction, molecular weight, amino acid composition and partial amino acid sequence. A high degree of similarity was observed among the proteins from these taxonomically distant bacterial species.

A new ribosomal proteinlocus in Escherichia coli: The gene for protein S6 maps at 97 min

SummaryA mutant of E. coli selected for temperaturesensitive growth on rich medium harbored an altered ribosomal protein S6 (Isono et al., 1976). This mutant was found to possess at least two mutations, one being responsible for the temperature-sensitivity and the other for the S6 alteration. Crosses with various Hfr strains as well as transductions with P lkc revealed that the former mutation mapped at 98 min and the latter at 97 min. Furthermore, recA derivatives of this mutant heteromerodiploid for this region possessed both the wild type and the mutant forms of S6. Thus it was established that the gene at 97 min was indeed the structural gene for protein S6 (rpsF) and not a gene modifying it.