Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

Summary Revertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.Paper No. 60 on Ribosomal Proteins. Preceding paper is by B. Wittmann-Liebold, Hoppe-Seyler's Z. physiol. Chemie, in press.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

Ribosomal proteins. 33. Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin

Ribosomal protein S12, the protein coded by the strA ciatron, was isolated from nine streptomycin-resistant mutants originating from various Escherichia coli strains. Analysis of the tryptic peptides revealed that each mutant had a single amino-acid replacement in one of two peptides: in mutants belonging to the allele types strA1, strA2 and strA60 the lysine residue in position 42 (peptide T6) of protein S12 is replaced by one of three ammo acids (asparagine, threonine or arginine) whereas the mutants belonging to allele type strA40 have a replacement of lysine by arginine in peptide T15. There is a good agreement between our protein-chemical data and earlier genetic data on streptomycin-resistant mutants.     

Properties of the interaction of ribosomal protein S4 and 16S RNA in Escherichia coli revertants from Streptomycin dependence to independence

Summary The binding properties of altered S4 proteins from E. coli revertants from streptomycin dependence to independence were investigated. Three of the proteins with the same length as the wild type protein, from mutants N424, N428 and N430, exhibited unchanged binding and conformational properties. However, three proteins with an altered length, from mutants N422, N425 and N433, bound more weakly to the 16S RNA, and their conformations were different from that of the wild type S4 protein. In the presence of the other 16S RNA binding proteins, no stimulation of the binding of the latter three proteins could be detected.Paper No. 40 on Ribosomal Proteins. Preceding paper is in press in Molec. gen. Genetics (1972).     

Ribosomal proteins. XXX. Specific protein binding sites on 23S RNA of Escherichia coli

Summary Strains of A. nidulans with a chromosome segment in duplicate (one in normal position, one translocated to another chromosome) are unstable at mitosis. During vegetative growth they produce variants which result from deletions in either of the duplicate segments.Caffeine increased the frequency of deletions from the duplicate segments of an unbalanced haploid a) without changing the proportions of the different deletion types and b) under conditions in which there were few, if any, induced breaks in the same segments of a balanced diploid. One possible explanation is that caffeine stimulates the mechanism which, in unbalanced strains, produces replication errors leading to deletions; an alternative is that it exposes the intrinsic instability of duplication strains by preventing the repair of spontaneous replication errors.     

Pleiotropic effects resulting from mutations in genes for ribosomal proteins: analysis of revertants from streptomycin dependence

In order to study the functions of the individual ribosomal proteins and their interaction, a group of revertants from streptomycin dependence to independence was analyzed. Reversion from dependence resulted from a number of different mutational events, all resulting in altered ribosome function. The mutants selected for study exhibited extensive pleiotropy—in addition to the elimination of the requirement for streptomycin for growth, the strains differed from the dependent parent and each other in growth rate, level of streptomycin resistance, effect of antibiotics on viability, rate of subunit assembly in vivo, affinity of isolated ribosomes for streptomycin and functionality of ribosomes in various cell-free assays.There appear to be strong correlations between the level of resistance to streptomycin in growing cells and the ability of the isolated ribosomes to bind streptomycin, the effect of antibiotic on cell-free protein synthesis programmed with natural message (but not poly(U)) and the degree of translational fidelity. There seems to be no relation between level of antibiotic resistance and the overall growth rate, the presence of a defect in ribosome assembly or the ribosomal protein altered by the mutation. Mutations in genes for 30 S proteins S4 and S5 can result in the same phenotype, while different changes in S4 in otherwise isogenic strains result in widely varying phenotypes.The wide variety of effects resulting from single mutational events suggests that each of these changes in a ribosomal protein changes the conformation of the ribosome or its ability to undergo configurational changes.     

Physical studies on the conformation of ribosomal protein S4 from Escherichia coli.

Proton magnetic resonance, circular dichroism and infrared spectroscopy were used to investigate the secondary and tertiary structure of the 16-S RNA binding protein S4 from Escherichia coli ribosomes. The proton magnetic resonance spectra of protein S4 in ribosomal reconstitution and low-salt buffers were identical and showed little dipolar broadening of the peaks, suggesting that the protein had an open extended structure. A ring-current-shifted apolar methyl resonance in the high-field region of the spectrum, together with a perturbation of the tyrosine ring proton resonance in the low-field region, indicated the existence of a specific tertiary fold in the polypeptide chain. This structure disappeared on lowering the pH below 5 or on heating above 30 degrees C, both processes being reversible. Circular dichroism measurements on protein S4 showed an alpha-helix content of 32% in reconstitution buffer compared with 26% in low-salt buffer. Heating the protein solution in reconstitution buffer above 35 degrees C reversibly disrupted this extra helix. Infrared studies on both solid films and solutions of protein S4 indicated the presence of little or no beta-structure. These results correlate well with the known RNA binding properties of protein S4.     

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

Summary 26 cold-resistant revertants of a cold-sensitiveEscherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered S8 than the original mutant and two revertants regained the wild-type form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant.The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is:aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....THe L30 mutation impairs growth and ribosomal assembly at 20°C and is therefore the first example of a mutant with a defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutants first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.     

Characterization of a temperature-sensitive Escherichia coli mutant and revertants with altered seryl-tRNA synthetase activity.

A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.