Eric R. Dabbs

Mutational alterations in 50 proteins of the Escherichia coli ribosome

SummaryA strain of Escherichia coli, VT, which spontaneously gives rise to mutations in many ribosomal proteins, has been used in conjunction with chemical mutagenesis and varying the subsequent incubation temperature to select mutants which have alterations in every ribosomal protein amenable to analysis of 70 S proteins on two-dimensional polyacrylamide gels under standard conditions. Alterations have been detected in 50 ribosomal proteins, namely in 20 from the small and in 30 from the large subunit. This is the most complete set of mutants with altered ribosomal proteins described so far. The difficulty until recently in obtaining mutations in most ribosomal proteins arises not because they are lethal, as has often been supposed, but because of the lack of a suitable selection heretofore.

A strain of Escherichia coli which gives rise to mutations in a large number of ribosomal proteins

SummaryA strain of E. coli K12 has been isolated which gives rise to mutations in a large number of ribosomal proteins. Mutant VT, which was derived from A19, shows a novel type of streptomycin dependence and has an altered ribosomal protein S8. Streptomycin-independent isolates from mutant VT contain a great variety of changed proteins on two-dimensional polyacrylamide gels. 120 revertants screened in this way have changes in thirteen 30S proteins and fifteen 50S proteins. Several mutants were found in which additional proteins are present on the ribosome. Further, there is one instance of a ribosomal protein (L1) being absent, and one of apparent doubling of a ribosomal protein (L7/12). The unique properties of mutant VT probably are the result of the altered S8.

Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

SummaryA battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.

Characterisation of a mutant from Escherichia coli lacking protein L15 and localisation of protein L15 by immuno-electron microscopy

SummaryTwo mutants lacking protein L15 from the ribosome as determined by two dimensional gels were investigated using a number of different immunological methods. One strain was found to possess several protein L15 moieties which differed in net charge and in size. The other showed no evidence of L15 cross-reacting material (CRM) on the ribosome or in the supernatant. Ribosomes of this strain were used as a control in the process of the localisation of protein L15 on the surface of the large subunit of Escherichia coli ribosomes. Antigenic determinants mapped in the angle between the central protuberance and the L1 protuberance. Protein L15 has been assigned a central role in the large subunit in vitro assembly map, in peptidyltransferase activity and in the binding of erythromycin, so the significance of a mutant lacking this protein is discussed.

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1. Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.

Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation

The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA). The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.

Assembly analysis of ribosomes from a mutant lacking the assembly-initiator protein L24: lack of L24 induces temperature sensitivity

SummaryPreviously, we have shown that the ribosomal protein L24 is one of two assembly-initiator proteins. L24 is essential for early steps of the assembly of the 50S ribosomal subunit but it is not involved in both the late assembly and the ribosomal functions. Surprisingly, an E. coli mutant (TA109-130) exists which lacks L24. This apparent paradox is analyzed and resolved in this paper. The phenotypic features of the mutant lacking L24, are a temperature sensitivity (growth severely reduced beyond 34° C), a very low growth rate already at permissive temperatures (at least six-fold slower than wild type) and an underproduction of 50S subunits (molar ratio of 30S to 50S about 1:0.5). The S value of the mutant large subunits is 47S, and they are normally active in poly(Phe) synthesis. The total protein of the mutant large subunits show negligible activity in the total reconstitution assay using the standard two-step procedure. Number analysis of the assembly-initiator proteins revealed that only one initiator protein is effective, as expected. The activity is restored upon addition of wild-type L24. However, when the temperature of the first step is lowered from 44° to 36° C, reconstitution of active particles occurs with a 50% efficiency in the absence of L24. The recovery of activity is accompanied by the appearance of again two initiator proteins, when the mutant TP50 lacking L24 is used in the reconstitution assay at the ‘permissive’ temperature of 36° C during the first step. These findings indicate that at least another protein or, alternatively, two other proteins take over the function of the assembly initiation at the lower temperature. Although the extent of the formation of active particles becomes independent of L24 below 36° C, the rate of formation is still strongly affected even at “permissive” temperatures. The presence of L24 reduces the activation energy of the rate-limiting step of the early assembly, i.e., the activation energy of RI50*(1) formation is 43±4 kcal/mol in the presence and 83±9 kcal in the absence of L24. The results presented provide an explanation of the phenotypic features of the mutant solely due to the assembly effects caused by the lack of L24.

A spectinomycin dependent mutant of Escherichia coli

SummaryA mutant of Escherichia coli B has been isolated which shows a novel phenotype of spectinomycin dependence. The mutant, termed RD, needs spectinomycin to grow at temperatures of 37° or below; it is unable to grow at 42° in either the presence or absence of spectinomycin. Secondary mutants which grow well in the absence of spectinomycin can be isolated spontaneously at a frequency of about 10-6. Two-dimensional gel electrophoresis of ribosomal proteins from 25 of these revertants showed that two revertants had an alteration in S4; one other showed an alteration in L5, and one showed an apparent absence of L1. Mutant RD itself had an altered less basic S5, which was maintained in all the revertants that were checked.Genetic analysis indicated that RD was a double mutant: one mutation, which alone conferred a spectinomycin resistant phenotype on the strain, was located in the strA region of the E. coli chromosome and was represented by the mutation in S5. The other mutation, which conferred the dependence on spectinomycin, mapped close to the rif locus.

A spontaneous mutant of Escherichia coli with protein L24 lacking from the ribosome

SummaryA spontaneous mutant that lacked ribosomal protein L24 was isolated and its derivatives investigated. The lesion responsible was close to, or in, rplX, the gene for protein L24. It led to a severe reduction in the amount of the large ribosomal subunit, even under permissive growth conditions. The mutation also led to a very slow growth rate and a temperature sensitive phenotype of carrier strains. Temperature indifferent secondary mutants frequently showed recovery of protein L24, but the protein was usually in a form larger than wildtype. Other secondary mutants had acquired an external suppressor that resulted in the simultaneous alteration of several other ribosomal proteins as well as the fractional presence of protein L24. Secondary mutants had normal amounts of the large ribosomal subunit, but it sedimented more slowly than normal.

The ribosomal components responsible for kasugamycin dependence, and its suppression, in a mutant of Escherichia coli

SummaryThe phenotype of a kasugamycin dependent mutant, MV17, was found to be the product of a kasugamycin resistance mutation in ksgA, together with a dependentizing mutation in rplW, the gene for large ribosomal subunit protein L23. Revertants from dependence on this small subunit targeted antibiotic were found to have mutational alterations in ribosomal proteins L23, L1, L11, and S9. The mutations causing alterations in L1 and L23 were shown to be responsible for the reversion and that altering L11 to be involved in the reversion.