Ribosomal proteins

Stöffler, Georg; Deusser, Ellen; Hg, Wittmann; Apirion, David

doi:10.1007/bf00569785

Cited by 67 publications

(12 citation statements)

References 15 publications

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“…Several such mutants are now available (32)(33)(34). We have found that some of these alterations, while not reflected in changes of electrophoretic properties, are detectable immunologically (33).…”

Section: Discussionmentioning

confidence: 84%

Sequence Differences of Escherichia coli 30S Ribosomal Proteins as Determined by Immunochemical Methods

Stöffler

Wittmann

1971

Proc. Natl. Acad. Sci. U.S.A.

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112

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Antisera specific for each of the 21 homogenous ribosomal proteins from 30S subunits of Escherichia coli were used to investigate, by immunodiffusion and immunoprecipitation, whether there are any extensive sequence homologies among these proteins.No immunological crossreactions were detected between any of the proteins. Therefore, we conclude that no significant common sequences exist among any of the 21 30S ribosomal proteins of E. coli.The exact features and structural requirements of antigenic determinants have been well characterized for synthetic antigens (1). Since the early studies of Landsteiner (2) on the specificity of serological reactions with natural protein antigens, progress has been made towards elucidating the properties of antigenic determinants in naturally occurring proteins (3, 4). The presence of many specific determinants on a single natural protein induces a heterogenous population of different antibodies, each specific for a different determinant distributed over the antigen protein (reviewed in ref. 5).Our purpose was to isolate antisera specific for each of the 30S proteins of Escherichia coli, so that we could investigate the extent of the structural differences between each protein.We considered this immunological method to be uniquely sensitive to structural details, and only surpassed by determination of the complete aminoacid sequence of each protein. The strategy of the experiments was to establish whether any immunological crossreaction occurred heterologously between individual homogenous 30S subunit proteins and their specific antisera. MATERIALS AND METHODSStrains. E. coli strain K12 (A19) (6) was used unless otherwise stated. E. coli B was used for the preparation of proteins S5 B and S7 B.Ribosomes and Ribosomal Proteins. Cells were grown in rich medium and harvested 2/3 of the way through the log phase. Ribosomes were obtained by differential centrifugation and washed twice with 0.5 M NH4Cl (7) or in 0.6 M (NH4)2SO4 (8). Subunits were separated and single 30S ribosomal proteins were isolated as described (9).The fractionated proteins were identified by 2-dimensional polyacrylamide gel electrophoresis (10, 11). 30-100 ,ug of each protein, together with 300-400 ,ug of total 70S ribosomal proteins, were electrophoresed; and the position of the main spot, relative to the faint background, identified the protein.Total 70S ribosomal protein used for immunochemical methods was prepared either by extraction with 67% acetic acid at 35 mM MgCl2 (8) or by incubating ribosomes with an equal volume of 8 M urea-4 M LiCl for 48 hr at 0C. The supernatant after a 30-min centrifugation was used. No differences were found between the two extraction methods, except that the proteins prepared with LiCl-urea were more soluble in buffers without urea. When total 30S ribosomal protein was used for serological work, the 30S particles were purified by two succeeding zonal centrifugations.Purity and Characterization of Proteins. The following methods were used to estimate the purity of each...

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Section: Discussionmentioning

confidence: 84%

Sequence Differences of Escherichia coli 30S Ribosomal Proteins as Determined by Immunochemical Methods

Stöffler

Wittmann

1971

Proc. Natl. Acad. Sci. U.S.A.

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112

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“…5 ) . By a combination of these methods most of the 50 S proteins could be purified to a purity of 95 -100 as shown by disc electrophoresis a t pH 4.5 in urea [IS], by dodecylsulfate gel electrophoresis [25], by twodimensional polyacrylamide gel electrophoresis [23] without added 70 S proteins as background and by cellulose acetate gel electrophoresis [26]. Proteins ( e .…”

Section: Resultsmentioning

confidence: 99%

Ribosomal Proteins

Hindennach

Kaltschmidt

Wittmann

1971

European Journal of Biochemistry

116

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50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM-cellulose chromatography. Gel filtration in Sephadex G-100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 950/, as shown by four methods. Proteins were isolated in yields of 6-90 mg depending mainly on the number and kind of purification steps.Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity.The protein moiety of ribosomes is very complex [I]. 30 S ribosomal subunits of Escherichia coli contain 21 proteins which were isolated and studied in various laboratories [ 2 -141. The number of ribosomal proteins in E . coli 50s subunits is about 50°/, higher than in 30 S subunits [8,10,15]. Because of the large number of 50 S proteins and their relatively similar chemical and physical properties, separation and isolation are very difficult and only possible by a combination of various methods which separate according to different criteria, e.g. protein charge and size as well as interaction of ribosomal proteins with each other and/or with ribosomal RNA in the particle.The present paper describes the isolation of 50s proteins in relatively large quantities (6-90 mg). The chemical, physical and immunological properties of the ribosomal proteins isolated by these methods are described elsewhere [I1 -14,271. MATERIAL AND METHODSIsolation of ribosomes and subunits from E . coli K12 (strain A19) and separation of proteins by CMcellulose and Sephadex G-I00 were described earlier [16]. Treatment of 50s subunits with LiCl followed previous procedures [17-221 with the following modifications.12g of 50s subunits were brought with 0.05M Tris-HC1 pH 7.8, 0.01 M Mg2+ to a concenbration of 10 mg/ml and dialyzed a t 4 "C for 4 h against 2 x 5 1 of 0.05 Tris-HC1 pH 7.8, 0.01 M Mg2+ and then for I2 h against 3 x 5 1 of the same buffer with 0.1 M Mg2+.One volume (= 1200ml) of 50s and one volume of cold 4 M LiC1, 0.1 M magnesium acetate, were mixed and stirred for 8 h in ice water. After centrifugation for 16 h a t 25000 rev./min in Spinco 42 rotors the supernatant (= UI) was dialyzed for 18 h against 4 x 5 1 100/, acetic acid plus 0.1 O/, mercaptoethanol.The sediment was resuspended in 1200 ml of cold 0.01 M Tris-HC1 pH 7.8, 0.1 M Mg2+, and dialyzed a t 4 "C for 4 h against 5 1 of this buffer and then for I2 h against 3 x 5 1 0.05 M Tris pH 7.8, 0.1 M Mg2+. The suspension was diluted 1 : 1 with cold 7 M LiC1, 0.1 M Mg2+, stirred in ice water for 8 h and centrifuged as described. The supernatant (= UII) was dialyzed as described for UI.The sediment was resuspended in 600ml cold distilled water. The...

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“…[l] to streptomycin independence is often accompanied by modification of proteins S4 [2-41 or S 5 [5,6]. Analysis of the properties of altered forms of S4 isolated from six revertants of this kind [7,8] showed (a) that in three cases S4 was modified without alteration of its chain length or of its binding to 16-S rRNA; (b) that in three other cases the C-terminal region of S4 was shortened or lengthened by up to 20 amino acids.…”

Section: -S Ribosomal Subunit Proteins Of An Escherichia Colimentioning

confidence: 99%

30‐S Ribosomal Subunit Proteins of an Escherichia coli Mutant in which Assembly of the Small Ribosomal Subunit Is Temperature‐Sensitive

Schmitt

Hayes

Reinbolt

1980

European Journal of Biochemistry

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Escherichiu coli 219ts2, a temperature-sensitive streptomycin-independent revertant of the streptomycin-dependent strain E. coli 209 is defective in 30-S ribosomal subunit assembly at 42 T. Total 30-S ribosomal subunit proteins of this strain contain two additional components one of which (a) migrates below and the other ( p ) to the right of protein S7 in two-dimensional polyacrylamide gel analyses carried out according to Kaltschniidt and Wittmann. These two components are also resolved from normal 30-S subunit proteins by chromatography on phosphocellulose. Tryptic fingerprinting of proteins a and / I ' identifies a as protein S7B, the form of S7 found in B strains of E. coli and fi as a mutant form of protein S4 produced by deletion of about 20 amino acids from the COOH terminus of the wild-type protein.Reversion of streptomycin-dependent Escherichiu coli mutants containing altered forms of protein S12[l] to streptomycin independence is often accompanied by modification of proteins S4 [2-41 or S 5 [5,6]. Analysis of the properties of altered forms of S4 isolated from six revertants of this kind [7,8] showed (a) that in three cases S4 was modified without alteration of its chain length or of its binding to 16-S rRNA; (b) that in three other cases the C-terminal region of S4 was shortened or lengthened by up to 20 amino acids. Alterations of the second type reduced the affinity of the mutant proteins for 16-S rRNA. Impaired binding of altered forms of S4 to 16-S rRNA may affect the binding of other ribosomal proteins and lead to abnormal assembly of ribosomal particles. The ribosomal protein phenotypes and physiological characteristics of a series of mutants of this type have been examined in detail by Olsson and Isaksson Rosset et al. [12] isolated a thermosensitive streptomycin-independent strain 219ts as a spontaneous revertant of the streptomycin-dependent strain E. coli 209 and studied its properties. At non-permissive temperatures synthesis of the 50-S ribosomal subunit is normal in E. coli 219ts, whereas that of the 30-S subunit is abnormal and yield particles with a sedimentation coefficient of about 28 S [13] which contain an immature (precursor) form of 16-S RNA [14].[9-11].Furthermore, the relative rates of synthesis of 50-S ribosomal subunit proteins in this mutant are unaltered at 42 ^C while those of several proteins of the 30-S subunit (SIO, S13, S20, S4 and S18) are changed [15].This paper describes the analysis of the proteins of 30-S ribosomal subunits synthesized at the permissive temperature (33 "C) by a second temperaturesensitive streptomycin-independent revertant of E. coli 209. This mutant, E. coli 219ts2, has a phenotype very similar to that of strain 219ts described by Rosset et al. [12].We show that reversion of strain 209 to strain 219ts2 is accompanied by reduction of the chain length of protein S4 and appearance in addition to S7K, the form of protein S7 characteristic of K strains of E. coli, of a second form of this protein with the electrophoretic properties of S7B. MATER...

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Ribosomal proteins

Cited by 67 publications

References 15 publications

Sequence Differences of Escherichia coli 30S Ribosomal Proteins as Determined by Immunochemical Methods

Sequence Differences of Escherichia coli 30S Ribosomal Proteins as Determined by Immunochemical Methods

Ribosomal Proteins

30‐S Ribosomal Subunit Proteins of an Escherichia coli Mutant in which Assembly of the Small Ribosomal Subunit Is Temperature‐Sensitive

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