Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation.
RNA Biol
; 18(12): 2363-2375, 2021 12.
Article
em En
| MEDLINE
| ID: mdl-33938388
ABSTRACT
Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Ribossomos
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Biossíntese de Proteínas
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Bioensaio
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RNA Mensageiro
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RNA de Transferência
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Aminoacil-RNA de Transferência
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Fatores de Alongamento de Peptídeos
Limite:
Humans
Idioma:
En
Revista:
RNA Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Suécia