Ribosomal Proteins of E. coli and Yeast

Kaltschmidt, E.; Rudloff, Viktor; Stöffler, Georg; Chersi, Alberto; Dzionara, M.; Donner, D.; Wittmann, H. G.

doi:10.1007/978-3-642-87534-2_2

Cited by 9 publications

(11 citation statements)

References 19 publications

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“…The sum of the amino acids of all peptides gives 120 amino acids for A-protein, which is in good agreement with the physical molecular weight [1,3]. I n earlier reports [34,36] the high a-helix content has been correlated with the relatively high alanine percentage in the A-protein.…”

Section: Discussionsupporting

confidence: 76%

50-S Ribsomal Proteins. Peptide Studies on Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Terhorst¹,

Wittmann-Liebold

Möller³

1972

Eur J Biochem

129

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Peptide studies on two acidic proteins, A, and A,, from 50-S ribosomes of Escherichia coli I. Amino acid compositions of the tryptic peptides of the two proteins are identical, with 2. The N-terminal amino acid sequence of A,-protein is Ser-Ile-Thr-Lys, while that of 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent 4. I n 500/, of the polypeptide chains, in both A,-or A,-protein, it specific lysine residue is indicate a closely related primary structure. exception of the amino terminal peptide.A,-protein is N-acetyl-Ser-Ile-Thr-Lys.with the physical molecular weight estimate.replaced by s-N-monomethyl-lysine.As reported in the preceding paper and their biological activity [S] which seems to be related to the phosphate energy liberation in the translocase step.The main objective of this study has been the precise determination of the difference in chemical structure between the two proteins. Thorough analysis of the tryptic peptides of the protein revealed that both have probably an identical primary structure and that the difference between the two is only confined to an acetyl group on the amino terminal end of the A,-protein, which is absent in A,-protein.From the peptide compositions the chemical molecular weight of the A-proteins have been estimated. MATERIALS AND METHODS Preparation of Tryptic PeptidesThe purification of A-protein, isolation and amino acid composition of A,-and A,-protein have been described elsewhere [i].Unusual Abbreviation. Dansyl, 1-dimethyl-aminonaphthalcne-5-sulphonyl.2 mg protein, determined by the microbiuret assay [l], was dissolved in 1 ml lo/, (NH,),CO, (pH 8.85) to which 20 p1 of a I mg/ml solution of trypsin treated with ~-l-tosyl-amido-2-phenylethylchloromethylketone, (Serva, Heidelberg, Germany) in 0.001 N HC1 was added to start the reaction at 37 "C. A second aliquot of enzyme (20 pg) was added after 1 h, the total digestion time being 4 h, after which the digest was freeze-dried, dissolved in 1 .O ml water and freeze-dried again. This residue was used directly for peptide mapping. Tryptic peptides for column chromatography were prepared by a 6-h digestion in dilute ammonia on a Radiometer pH-stat a t p H 8.0,37 "C. The enzyme to substrate weight ratio was also 1 to 50. The mixture was then frozen and lyophilized once. Separation of the Tryptic PeptidesPeptide maps were made essentially according to Clegg et al. [9]. High-voltage paper electrophoresis on Whatmann 3-MM paper was performed in a "Varso1"-cooled tank (Savant Instruments, Hicksville, U.S.A.) at 30 V/cm for 3 h with a buffer (pH 4.7) of pyridine-acetic acid-water (5:5: 390, by vol.). The solvent for paper chromatography was n-butanol-acetic acid-water-pyridine (15:3: 12: 10, by vol.) (Hills solvent). Peptides were detected by staining the paper with a 0.050/, ninhydrin-acetone solution a t 40 "C for 30 min. The coloured spots were subsequently eluted from the paper with ZOO/, pyridine in water.

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Section: Discussionsupporting

confidence: 76%

50-S Ribsomal Proteins. Peptide Studies on Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Terhorst¹,

Wittmann-Liebold

Möller³

1972

Eur J Biochem

129

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“…If [21]. The identification of the purified proteins was done by two-dimensional electrophoresis [22] (Fig. 2) and by double immunodiffusion tests [l 11.…”

Section: Resultsmentioning

confidence: 99%

“…The isolated proteins corresponded to proteins Sl -S21 as identified by two-dimensional electrophoresis of the protein moiety of 30 S subunits [22]. Two proteins (S3 and SII) need some discussion.…”

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confidence: 99%

Ribosomal Proteins

Hindennach

Stöffler

Wittmann

1971

European Journal of Biochemistry

181

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All 21 ribosomal proteins from 3 0 s subunits of Escheriehia coli were isolated in a pure state by the following methods: separation of subunits by zonal centrifugation in B XV rotors, extraction of proteins, CM-cellulose chromatography with pyridine formate gradients, gel filtration on Sephadex G-100 and, in some cases, preparative polyacrylamide block electrophoresis. The purity of isolated proteins was 97-100°/, shown by disc electrophoresis a t pH 4.5 in urea, by two-dimensional polyacrylamide electrophoresis, by dodecylsulfate gel electrophoresis and by cellulose acetate gel electrophoresis. The yield of the isolated proteins was 7 -110 mg depending on the protein and the number of purification steps.Fractionation of total 3 0 s proteins with ammonium sulfate is recommended for quick isolation of proteins S15 and 520 in large quantities.Waller [ 11 showed that starch gel electrophoresis separates the protein moiety of Escherichia eoli ribosomes into many bands. Later studies [2-101 demonstrated that the bands correspond to proteins of different physical and chemical structure. There exists also no immunological cross reaction between the individual 30 S proteins [ 111. There is agreement between various groups on the number, namely 21, and on the nomenclature of the ribosomal proteins from the 3 0 s subunits of E. coli [12].Good and reproducible methods for the isolation of ribosomal proteins on a big scale are the prerequisite not only for their chemical and physical characterization but also for protein-chemical studies on their amino sequences. The present paper describes t h e isolation of all 30 S ribosomal proteins of E. eoli in relatively large quantities (7-110 mg) and high purity (97-100°/,). Haan, Germany). After removal of cell debris and alcoa a t 16000 rev./min for 40 min in a Servall SS-34 rotor, ribosomes were sedimented by overnight centrifugation a t 25000rev.lmin in a Spinco 42 rotor. Ribosomes were resuspended in the buffer described above and purified by three cycles of differential centrifugation, the last with a buffer containing 0.5 M NH,C1 in 0.02 M Tris-HC1 pH 7.8,0.002 M MgC1, and 0.01 M mercaptoethanol. For our first isolations of ribosomes a buffer [14] containing 0.6 M (NH,),SO, instead of 0.5MNH4C1 was used for the last two cycles. MATERIAL AND METHODS IsolationFor separation of subunits aliquots of about 1.5 g ribosomes in 30 ml were dialyzed overnight against 5 1 of 0.01 M potassium phosphate pH 7.4. 0.001 hl MgCl, and 0.01 M mercaptoethanol and centrifuged in Spinco B XV Ti zonal rotors for 17 h a t 25000 rev./ min. A gradient of 7.5O/, to 38O/, sucrose in phosphate buffer and 600 ml overlay was used [15]. Absorbance was recorded a t 295 nm in a flow cell of a Beckman DB spectrophotometer. Subunits were precipitated either with ammonium sulfate (2/3 saturation a t 4 "C)

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“…This was performed by the method of KaltSchmidt and Wittmann [21]. The LiCl split proteins from the 50-S subunits were run on two-dimensional polyacrylamide gels.…”

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 99%

An Attempt at the Identification of the Proteins Involved in the Incorporation of 5‐S RNA during 50‐S Ribosomal Subunit Assembly

Gray

Garrett

Stöffler

et al. 1972

European Journal of Biochemistry

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I n an attempt to determine the RNA-protein interactions involved in the biogenesis of the 50-S ribosomal subunit we have developed a simple and specific system in vitro which reflects some of the intricacies of the Escherichia coli 50-5 subunit. By studying the interaction of isolated ribosomal proteins with 5-5 and 23-S RNAs we have demonstrated the formation of a stable complex which contains 5-S RNA and 23-S RNA and which is dependent upon only two or three ribosomal proteins. The characteristics of this complex have been determined and the responsible proteins identified. The 5-5 -23-5 RNA complex is dependent upon protein L18 in conjunction with either L6 or L25. Although it would be premature to conclude that these results in vitro are directly related to the assembly map in vivo of the 50-S subunit, there is a strong correlation between our results and those obtained with various natural and artificial ribosomal subparticles.The interactions occurring between ribosomal proteins and ribosomal RNA within the intact ribosome appear to be quite complicated and involve linear as well as tridimensional structural information on the part of the RNA. Since the sequential reconstitution of the Escherichia coli 30-5 ribosomal subunit in vitro [l], there has been considerable progress made in understanding some of the RNA-protein interactions involved in both the structure and function offhe 30-S subunit [2--61; however, the larger 50-5 ribosomal subunit of E . coli which contains approximately 34 proteins and two RNA molecules [S], presents a larger problem.There are a few recent experimental reports which discuss the topography and arrangement of the 50-5 ribosomal proteins and RNA while in the complete particle. Most of these studies with E. coli have used either controlled release or partial digestion of the ribosomal proteins [7-91. Studies on the location of ribosomal RNAs within the subunit have proved equally difficult, although one laboratory has tried to localize the protein-binding sites on the 23-S RNA by partial degradation of the 50-S subunit into two fragment particles by cleavage of the 23-5 RNA, and subsequent deterDefinition. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm path-length cell. mination of the protein content of the isolated fragment particles [lo].Another approach to the RNA-protein problem, and the one which we have chosen, is the study of direct interaction of isolated proteins with purified RNAs. It has been shown, using a oneprotein-one-RNA system, that there are only eight 50-5 proteins which are able to bind independently and in equimolar amounts to 23-5 RNA [ll]. These very simple complexes could therefore aid in determining the precise RNA sequences involved in protein-RNA recognition. However, the functionally complete 50-5 contains 5-5 RNA as well and it would be advantageous to study the direct interactions between both RNAs and any proteins necessary for the formation of a complex containi...

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Ribosomal Proteins of E. coli and Yeast

Cited by 9 publications

References 19 publications

50-S Ribsomal Proteins. Peptide Studies on Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

50-S Ribsomal Proteins. Peptide Studies on Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Ribosomal Proteins

An Attempt at the Identification of the Proteins Involved in the Incorporation of 5‐S RNA during 50‐S Ribosomal Subunit Assembly

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