RESUMO
Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site binding codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5'-termini that could predetermine the position of the tRNA(Phe) on the ribosome by the location of P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3' of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide-induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2-127) and/or in the C-terminal fragment 190-236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.
Assuntos
Códon/química , Oligopeptídeos/química , Elongação Traducional da Cadeia Peptídica/fisiologia , Terminação Traducional da Cadeia Peptídica/fisiologia , Proteínas Ribossômicas/química , Códon/metabolismo , Humanos , Oligopeptídeos/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos , Raios UltravioletaAssuntos
Genes MDR/genética , RNA de Cadeia Dupla/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Sequência de Bases , Expressão Gênica , Humanos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences. The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5' ends and were complementary to 18S rRNA sequences 609-618 and 1047-1061, homologous to the highly conserved regions designated as the "530 stem-loop" and "790 stem-loop", respectively, in Escherichia coli 16S rRNA. Ribosomal proteins surrounding these sequences were the main targets of the cross-linking. Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609-618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047-1061. Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNA(Phe).
