Determination of the Complete Amino-Acid Sequence of Protein S4 from Escherichia coli Ribosomes

Schiltz, Emil; Reinbolt, Joseph

doi:10.1111/j.1432-1033.1975.tb02253.x

Cited by 54 publications

(29 citation statements)

References 48 publications

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“…The tryptic fingerprint of protein B (Fig. 4B) is very similar to that of protein S4 from the wild type [28]. The major difference is the presence of an additional peptide Tx (arrow on Fig.…”

Section: Identification Of Proteins C( and Pmentioning

confidence: 58%

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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Section: Identification Of Proteins C( and Pmentioning

confidence: 58%

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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“…These fingerprints are illustrated on figs.3 and 4. The position of the tryptic peptides was assigned from the fingerprint on paper described by Schiltz and Reinbolt [28] and from some information kindly provided by Dr Yaguchi, Berlin. Comparison of the peptide maps revealed differences in some ninhydrinstained spots.…”

Section: Resultsmentioning

confidence: 99%

Studies of the binding of E. coli ribosomal protein S4 to 16S rRNA after UV irradiation of the S4—16S rRNA complex

1975

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“…The protein S4 has a cysteine residue at position 31 from the N-terminus [21], and this cysteine SH group is reactive to N-ethylmaleimide [6]. The maximum linear length of the bromopyruvdte linker is about 0.6 nm.…”

Section: Discussionmentioning

confidence: 99%

“…Covalent cross-linking between specific groups in macromolecules is helpful for establishing neighborhoods within each biological system. Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine [l, 21. The reaction is specific for singlestranded regions of nucleic acids and converts cytosine into N4-aminocytosine.…”

mentioning

confidence: 99%

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

et al. 1986

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Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-0x0 group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30s subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgClz at pH 7.0 and 37°C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16s rRNA were derivatized into N4-aminocytosines. 35S-labeled 30s subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37°C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The cosedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30s subunits. The protein portion was labeled with 1251, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.Chemical cross-linking between biological macromolecules is a useful technique for studying spacial arrangements in multi-component systems such as organellas and complex assemblies of biomolecules. Covalent cross-linking between specific groups in macromolecules is helpful for establishing neighborhoods within each biological system. Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine [l, 21. The reaction is specific for singlestranded regions of nucleic acids and converts cytosine into N4-aminocytosine. The hydrazino group at position 4 of this product was shown to react with ketones, aldehydes and imidates [3]. Bromopyruvate has been used for the modification of protein SH groups [4]. Therefore, the use of bromopyruvate after the bisulfite/hydrazine modification would allow linking between N4-aminocytosine and a protein SH group. This method of cross-linking has several advantages: the reactions are carried out in nearly neutral solutions, the residues being cross-linked are specific, the mechanism is established, and all the reagents are watersoluble and commercially available. We have shown that cross-linking of poly(C) with glutathione can indeed be accomplished by use of this two-step reaction [5]. The Escherichia coli ribosomal 30s subunit is composed of 16s rRNA and 21 single proteins of different kinds. When the subunits a...

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Determination of the Complete Amino-Acid Sequence of Protein S4 from Escherichia coli Ribosomes

Cited by 54 publications

References 48 publications

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

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

Studies of the binding of E. coli ribosomal protein S4 to 16S rRNA after UV irradiation of the S4—16S rRNA complex

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

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