Biochemical and Biophysical Research Communications
A phage RNA-binding protein binds to a non-cognate structured RNA and stabilizes its core structure
Section snippets
Materials and methods
RNA and protein preparation. Preparation and radioactive labeling of the Candida group I intron ribozyme RNAs were performed as described [17]. MBP and MS2–MBP fusion protein were expressed and purified [19].
Analysis of the ribozyme activity. The purified RNAs randomly labeled by incorporation of [α-32P]GTP during in vitro transcription was denatured and equilibrated as previously described [17]. The equilibrated RNA was incubated in the folding buffer containing 2 mM or 6 mM MgCl2 and 1 μM of the
MS2 CP binds to the Candida group I ribozyme
The binding capacity of MS2 CP with the Candida ribozyme was investigated by gel retardation assay, showing that the level of the MS2–ribozyme complex increased with the concentrations of MS2 protein (Fig. 1A). Quantification of three independent sets of experiments resulted in a Kd value of 610 ± 130 nM (Fig. 1B). Consistently, a Kd value of 226 ± 30 nM was obtained using fluorescence spectrum assay (Supplementary Figure S1). These data suggested that MS2 CP binds to ribozyme RNA at an affinity much
Discussion
We have demonstrated that MS2 CP binds to a eukaryotic group I ribozyme, which alters the ribozyme 3′ hydrolysis activity. A new method developed in this report showed that MS2 primarily interacts with the P5ab peripheral structure of the ribozyme. T1 footprinting and CLiPE (UV cross-linking coupled with primer extension) assays showed that MS2 binding stabilizes the L9–P5 tertiary interaction and leads to a more compact ribozyme core important for the 3′ hydrolysis activity of the ribozyme.
Acknowledgments
We thank R. Reed (Harvard University, Cambridge, MA) for generously giving us the plasmid encoding the MS2–MBP fusion protein. This work is supported by National Science Foundation of China (30330170) and by the National Basic Research Program (973) of China through Grant 2005CB724604 awarded to Y. Zhang.
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