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Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19
Author(s) -
Koukiekolo Roger,
Sagan Selena M.,
Pezacki John Paul
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.05.064
Subject(s) - small interfering rna , gene silencing , rna , chemistry , binding site , rna silencing , rna binding protein , histidine , biochemistry , microbiology and biotechnology , ligand binding assay , biophysics , rna interference , biology , amino acid , receptor , gene
The RNA silencing pathway is an important component of the anti‐viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19 kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short‐interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence‐based method for assaying CIRV p19:siRNA binding using Ni 2+ –NTA coated 96‐well plates. Using this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH‐dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent p K a values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6.