Ribosomes from the following strains of methanogenic bacteria were isolated and their protein patterns analysed by twodimensional polyacrylamide gel electrophoresis: Methano-sarcina (Ms.) barkeri (DSM 800); Ms. barkeri (morphotype II) (DSM 1232); Methanococcus (Mc.) vannielii (DSM 1224); Methanobacterium (Mb.) thermoautotrophicum (DSM 1053); Mb. arbophilicum (DSM 1125); Mb. formicicum and Mb. strain M. o. H. Ribosomes from Ms. barkeri were analysed in more detail. They possess an apparent sedimentation constant of 70S and dissociate at 1 mM Mg++ into 30S and 50S subunits. Electropherograms of 30 S subunits purified twice on sucrose gradients exhibit at least 27 protein spots, those for 50S subunits at least 33. The individuality of these spots has not yet rigorously been determined. The electrophoretic pattern of the two strains of Ms. barkeri investigated differ with respect to a considerable number of proteins, which indicates a high degree of diversity even within one morphologically similar group.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAlteration of ribosomal protein L6 in gentamicin-resistant strains of Escherichia coli. Effects on fidelity of protein synthesisRobert Kuehberger, Wolfgang Piepersberg, Angelika Petzet, Peter Buckel, and August BoeckCite this: Biochemistry 1979, 18, 1, 187–193Publication Date (Print):January 1, 1979Publication History Published online1 May 2002Published inissue 1 January 1979https://doi.org/10.1021/bi00568a028RIGHTS & PERMISSIONSArticle Views172Altmetric-Citations69LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

FEBS LettersVolume 104, Issue 2 p. 317-321 Full-length articleFree Access Ribosomal ambiguity (ram) mutations facilitate dihydrostreptomycin binding to ribosomes August Böck, August Böck Lehrstuhl für Mikrobiologie der Universität Regensburg, 8400 Regensburg, GermanySearch for more papers by this authorAngelika Petzet, Angelika Petzet Lehrstuhl für Mikrobiologie der Universität Regensburg, 8400 Regensburg, GermanySearch for more papers by this authorWolfgang Piepersberg, Wolfgang Piepersberg Lehrstuhl für Mikrobiologie der Universität Regensburg, 8400 Regensburg, GermanySearch for more papers by this author August Böck, August Böck Lehrstuhl für Mikrobiologie der Universität Regensburg, 8400 Regensburg, GermanySearch for more papers by this authorAngelika Petzet, Angelika Petzet Lehrstuhl für Mikrobiologie der Universität Regensburg, 8400 Regensburg, GermanySearch for more papers by this authorWolfgang Piepersberg, Wolfgang Piepersberg Lehrstuhl für Mikrobiologie der Universität Regensburg, 8400 Regensburg, GermanySearch for more papers by this author First published: August 15, 1979 https://doi.org/10.1016/0014-5793(79)80842-XCitations: 27AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume104, Issue2August 15, 1979Pages 317-321 ReferencesRelatedInformation

High level lincomycin resistant strains of Escherichia coli were isolated and screened for altered ribosomal proteins and functions. Amongst 58 strains investigated by electrophoresis one had an altered ribosomal protein S7, another one a mutated L14 and two showed altered L15 proteins.

A set of mutants affected in translational fidelity was constructed by transduction within an otherwise isogenic Escherichia coli B argF40 argR11 background. Alterations in ribosomal proteins S4, S5, S12 and L6 either as single mutations or in various combinations were compared for their effects on aminoglycoside phenotypes, on in vivo and in vitro mis-reading and on the rate of peptide bond formation. Results may be summarized as follows: (i) Strains carrying two ambiguity mutations on the ribosome without any restrictive mutation are viable. When together, they only weakly increase the level of mistranslation as judged by several in vivo and in vitro test systems. (ii) The combination of two ram mutations causes a very strong cooperative increase of streptomycin sensitivity, irrespective of whether the strains have a wild-type S12 or mutationally altered S12 proteins (of the drug-resistant or-dependent types) on their ribosomes; (iii) The S4 and S5 ram mutations do not alter the response of the ribosome to aminoglycosides of the 2-desoxystreptamine group which are structurally unrelated to streptomycin. This is interpreted in terms of an effect of these ram mutations on the streptomycin binding site but not on the site(s) of binding of the other aminoglycosides. (iv) The rate of polypeptide bond formation which was determined from the kinetics of β-galactosidase induction is not significantly changed in strains bearing the ram and the strA (streptomycin-resistant) alleles. In contrast, the L6 and the strA (streptomycin-dependent) alleles strongly reduce the rate of polypeptide elongation which mechanistically might be connected with restriction of ambiguity (Ninio, 1974) in these cases.

Spontaneous and ethylmethane-sulfonate induced mutants of Escherichia coli resistant to gentamicin sulfate were isolated and investigated for alterations in the ribosomal protein pattern. It was found by two-dimensional polyacrylamide gel electrophoresis that three independently isolated strains did not show any spot for ribosomal protein L6. On cochromatography of radioactively labelled mutant and wild-type ribosomal proteins on carboxymethyl-cellulose columns a shift of the elution position of protein L6 was observed, the new elution positions being characteristic for the individual mutants analyzed which indicates that they possess different alterations in the L6 primary structure.

A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes. The transducing lambda phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (Mr 78,000) is still unidentified.

The effect of three different types of mutations in ribosomal protein S5 of Escherichia coli on translational fidelity has been studied. Two of them, namely that conferring resistance to spectinomycin and that selected for partial suppression of a temperaturesensitive alanyl-tRNA synthetase mutation, do not exhibit ribosomal ambiguity in the in vivo and in vitro test system employed. In constrast, mutations in ribosomal protein S5 selected for suppression of streptomycin dependence mutations are able to derestrict the restriction of translational ambiguity imposed by strA mutations, though to different degrees depending on the type of mutation. Mutants in which streptomycin dependence is suppressed by an alteration in protein S5 are more restrictive than mutants resistant to streptomycin. Again, the extent of restriction depends on the type of the strAd allele.

Three different phenylalanyl‐tRNA synthetases have been purified to near homogeneity, one from a wild‐type strain of Escherichia coli and the others from two independently isolated p‐fluorophenylalanine‐resistant strains. The mutant enzymes were not able to use p‐fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild‐type enzyme by several electrophoretic and immunological criteria.The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl‐tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild‐type and mutant subunits indicate that the mutant α subunit is responsible for p‐fluorophenylalanine resistance and therefore seems to carry the phenylalanine‐binding site or to participate in its formation.

In order to obtain E. coli strains altered in ribosomal proteins the following isolation technique was used: Phage P1 grown in a streptomycin resistant E. coli strain, was mutagenized by hydroxylamine or nitrous acid, and was used to transduce into a strain auxotrophic for aroE. Transductants with streptomycin resistance and aroE prototrophy were selected and tested for their growth at various temperatures (20°, 30° and 42°) and their response to different antibiotics.

The molecular weight of l‐phenylalanyl‐tRNA synthetase was redetermined. A new value of 267 000 was obtained by high‐speed analytical ultracentrifugation and Sephadex G‐200 gel chromatography. The value is in contrast to the molecular weight of 180000 determined previouslyFrom the molecular weight 267000 and the sedimentation coefficient s20,w= 8.6 S the ratio f/f0= 1.67 was calculated. The magnitude of f/f0 is in accord with the molecular weight 183000 obtained from sucrose gradient centrifugation. The results indicate that the enzyme is highly hydrated and/or assumes the shape of a rod or a disc.The subunit composition was reinvestigated by sodium dodecylsulfate gel electrophoresis, guanidinium chloride gel filtration and by reactivation experiments of pH‐2 inactivated enzyme. The results are consistent with a subunit structure of the α2β2 type. Molecular weights of the subunits are 39000 and 94000, respectively.

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.

The genetic location has been determined of two mutations which suppress the temperature-sensitive phenotype of an alanyl-tRNA-synthetase mutant of Escherichia coli and which are correlated with alterations of the ribosomal protein S20. Both mutations map at the same chromosomal site; the gene order relative to other markers of the Escherichia coli map is thr-sup-pyrA-araC-leu.

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C 6 substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity ( gnd − ) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase ( pgi − ) restores the ability of these fda − gnd − strains to grow on glucose at 40 C. Glucose metabolism of these fda − gnd − pgi − strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l -alanine. Predominant labeling of the carboxyl-carbon of l -alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.

The stoichiometry of substrate binding to phenylalanyl‐tRNA synthetase from Escherichia coli K10 was investigated. Isolation of the phenylalanyl · adenylate · enzyme complex by means of gel filtration revealed that phenylalanine and the nucleotide are bound in about equimolar amounts and in a stoichiometric ratio. Stable complexes between tRNA Phe and phenylalanyl‐tRNA synthetase could be separated from free tRNA Phe via sucrose‐gradient zone‐centrifugation or gel filtration. Determination of the amount of tRNA Phe bound to phenylalanyl‐tRNA synthetase (molecular weight 181000) by absorption at 260 nm or by the amino‐acid‐acceptance activity yielded ratios close to one. tRNA Phe · enzyme complexes were most stable at pH values below 6.5; complex formation between phenylalanyl‐tRNA synthetase and non‐cognate tRNA species was not observed. For determination of the affinity and ratio of binding of phenylalanine the non‐equilibrium dialysis method was employed: a K d of 30 μM was obtained; by the criteria of this method one binding site for the amino acid could be demonstrated. Phenylalanyl‐tRNA synthetase of E. coli is protected against heat inactivation by Mg, ATP, ATP and phenylalanine or tRNA Phe . By means of the protection constant, the apparent affinity constant for tRNA Phe was determined to be 3 × 10 7 M −1 at pH 6.0 and 37 °C. The dependence of the π‐values obtained on pH indicates that the affinity of phenylalanyl‐tRNA synthetase for tRNA drastically increases at more acid pH. The same tendency was obtained upon determination of the pH‐dependence of the K m values for tRNA Phe . Out of the 24 SH‐groups of phenylalanyl‐tRNA synthetase which are reacting with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) in the presence of 8 M urea only less than one half of them are oxidized on the native enzyme. The kinetics of titration of the sulfhydryl is grossly changed in the presence of ATP, ATP and phenylalanine or tRNA Phe . In addition, in the presence of ATP and phenylalanine or of tRNA Phe 2‐3 SH‐groups are completely protected from oxidation. Under this condition loss of activity which accompanies the SH‐group oxidation is greatly diminished.

Fingerprints of tryptic digests of phenylalanyl‐tRNA synthetase of Escherichia coli NP 3 suggest that the smallest identical subunit is one fourth of the size of the native protein (mol. wt. 181000). Determination of the subunit size by gel electrophoresis reveals a molecular weight of 43000 ± 2000. By the criteria employed the enzyme therefore seems to be composed of four identical subunits.Phenylalanyl‐tRNA synthetase which has been denatured by treatment with 8 M urea is able to reform active enzyme indistinguishable by its size from the native protein. Again, a size of about 45000 was determined for the subunits active in the association reaction.

Complementation in vitro has been studied between the phenylalanyl‐tRNA synthetase subunits present in cell‐free extracts of temperature‐sensitive mutants of Escherichia coli. These mutant subunits, which judged from their size are dimers, form a hybrid enzyme with the dissociation products present in urea treated wild type enzyme preparations. The enzymatically active complementation product possesses the same size as the native enzyme but differs from it in its higher heat sensitivity. Complementation between the phenylalanyl‐tRNA synthetase cross‐reacting material of the different mutants alone could not be demonstrated under the conditions tested.

Böck, August(Purdue University, Lafayette, Ind.),and Frederick C. Neidhardt. Properties of a mutant ofEscherichia coliwith a temperature-sensitive fructose-1,6-diphosphate aldolase. J. Bacteriol.92:470–476. 1966.—A mutant ofEscherichia coliin which fructose-1,6-diphosphate aldolase functions at 30 C but not at 40 C was used to study the physiological effect of a specific block in the Embden-Meyerhof glycolytic pathway. Growth of the mutant at 40 C was found to be inhibited by the presence of glucose or certain related compounds in the medium. At 40 C, glucose was metabolized at 30 to 40% of the control rate and was abnormal in that glucose was converted into other six-carbon substances (probably gluconate, in large part) that were released into the culture medium. The inhibition was complete, but transient; its duration depended upon the initial amount of inhibitor added. The resumption of growth at 40 C was correlated with the further catabolism of the excreted compounds. When glycerol was used to grow the mutant at 40 C, the growth inhibition by glucose was accompanied by cessation of glycerol metabolism. Growth on α-glycerol phosphate was not inhibited under these conditions, implicating glycerol kinase as a possible site of inhibition; no inhibition of glycerol kinase by sugar phosphates, however, could be detected in vitro. The inhibitory effect of glucose on growth at 40 C is not caused by a deficit of intracellular adenosine triphosphate, but may be the result of a generalized poisoning of many cell processes by a greatly increased intracellular concentration of fructose-1,6-diphosphate, the substrate of the damaged enzyme.