RESUMO
The cleavable homobifunctional reagent dichloro[N,N,N',N'-tetrakis(2-aminoethyl)-1,6-hexamethylenediamminedi platinum (II)] dichloride was used for studying rRNA-protein cross-links in free 35S-labelled 70 S ribosomes and within initiation complex ribosome.AUGU6.fMet-tRNA(fMet). It was shown that the sets of proteins cross-linked to 16 S and 23 S rRNA in free 70 S ribosomes and in 70 S initiation complex do not differ significantly. The authors are the first to demonstrate most of the 23 S rRNA-protein cross-links and some 16 S rRNA-protein cross-links, in particular those with L7/L12 protein.
Assuntos
Proteínas de Bactérias/metabolismo , Reagentes de Ligações Cruzadas , Compostos Organoplatínicos , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismoRESUMO
rRNA-protein cross-links in free E. coli 35S-labeled 70 S ribosomes and in the initiation complex 35S-labeled 70 S ribosome.AUGU6.fMet-tRNA(fMet) were studied with the aid of a new type of binuclear Pt(II) compound - dichlorotetra-ammine(1,6-hexamethylenediaminediplatinum++ +) dichloride. The use of this reagent allowed us to reveal differences in the rRNA-protein neighbourhood in free 70 S ribosomes and in the initiation complex. Proteins L3, L6, L23 and L25 were shown to cross-link to 23 S rRNA only in the initiation complex, whereas proteins L1, L13, L14, L16, L17, L18, L22, L28 and S1 did so in both free ribosomes and the complex. 16 S rRNA was found to be cross-linked preferentially to a single protein, S1, in both states of the ribosomes.
Assuntos
Escherichia coli/metabolismo , Compostos Organoplatínicos/farmacologia , RNA Ribossômico 23S/metabolismo , RNA Ribossômico/metabolismo , RNA de Transferência de Metionina , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Centrifugação com Gradiente de Concentração , Reagentes de Ligações Cruzadas , Eletroforese em Gel Bidimensional , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Tioureia/farmacologiaRESUMO
A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.