RESUMO
Affinity labelling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]benzylidene derivative of AUGU6 was studied within the initiation complex (complex I) obtained by using fMet-tRNAMetf and initiation factors and within the pretranslocational complex (complex II) obtained by treatment of complex I with the ternary complex Phe-tRNAPhe.GTP.EF-Tu. Both proteins and rRNA of 30 S as well as 50 S subunits were found to be labelled. Sets of proteins labelled within complexes I and II differ considerably. Within complex II, proteins S13 and L10 were labelled preferentially. On the other hand, within complex I, multiple modification is observed (proteins S4, S12, S13, S14, S15, S18, S19, S20/L26 were found to be alkylated) despite the single fixation of a template in the ribosome by interaction of the AUG codon with fMet-tRNAMetf.
Assuntos
Marcadores de Afinidade/metabolismo , Códon , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro , Ribossomos/metabolismo , Sítios de Ligação , Complexo II de Transporte de Elétrons , Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Complexos Multienzimáticos/metabolismo , Compostos de Mostarda , NAD(P)H Desidrogenase (Quinona) , Oxirredutases/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Quinona Redutases/metabolismo , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo , Succinato Desidrogenase/metabolismoRESUMO
Hexaribouridylic acid, prepared by digestion of poly(U) with cobra venom endonuclease, and trinucleotide AUG synthesized chemically by triester approach were joined by RNA-ligase to yield a nonaribonucleotide AUGU6 bearing the initiation codon at its 5'-terminus. 2',3'-O-(4-[N-(2-chloro(or hydroxy) ethyl-N-Methylamino])- benzylidene residues were introduced at the 3'-terminus of oligonucleotide AUGU6 and its benzylidene derivatives AUGU6CHRCl or AUGU6CHROH were obtained. The mRNA analogs synthesized were tested for their template activity in the formation of 70S initiation complex. AUGU6, AUGU6CHRC1 and AUGU6CHROH were shown to stimulate factor-dependent binding of fMet-tRNA to ribosomes. The effect of benzylidene fragment on the template activity of AUGU6CHROH in the course of translation process was studied. It was shown that AUGU6CHROH stimulates synthesis of di- and tripeptides with the same efficiency as AUGU6.
