Quantitative Analysis of RNA Chaperone Activity by Native Gel Electrophoresis and Fluorescence Spectroscopy.
Methods Mol Biol
; 2106: 19-39, 2020.
Article
em En
| MEDLINE
| ID: mdl-31889249
ABSTRACT
Diverse types of RNA-binding proteins chaperone the interactions of noncoding RNAs by increasing the rate of RNA base pairing and by stabilizing the final RNA duplex. The E. coli protein Hfq facilitates interactions between small noncoding RNAs and their target mRNAs. The chaperone and RNA annealing activity of Hfq and other RNA chaperones can be evaluated by determining the kinetics of RNA base pairing in the presence and absence of the protein. This chapter presents protocols for measuring RNA annealing kinetics using electrophoretic gel mobility shift assays (EMSA), stopped-flow fluorescence, and fluorescence anisotropy. EMSA is low cost and can resolve reaction intermediates of natural small RNAs and mRNA fragments, as long as the complexes are sufficiently long-lived (≥10 s) to be trapped during electrophoresis. Stopped-flow fluorescence can detect annealing reactions between 1 ms and 30 s and is best suited for measuring the rapid annealing of oligoribonucleotides. Fluorescence anisotropy reports the physical size of the complex and is well-suited for monitoring the association and dissociation of RNA from Hfq during the chaperone cycle.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Chaperonas Moleculares
/
Ensaio de Desvio de Mobilidade Eletroforética
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article