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32P RNA and quantified with a Phosphorlmager (Molecular Dynamics).
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4243202409
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-kt), where B is the size of the burst, if any, and A + B gives the endpoint.
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Splicing assays were performed as described (4) To measure the CBP2 association rate, CBP2 and pG were added simultaneously to trace concentrations of renatured RNA. For experiments designed to measure the rate of conversion of the folded nbozyme, CBP2 was incubated with RNA for 1 hour to ensure complete binding prior to initiating splicing by addition of pG (final concentration 2 mM)
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16
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4243089070
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note
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If the CBP2 concentration is low enough, some step involving the protein will become rate-limiting. However, this concentration must be much smaller than 0.2 nM, the lowest concentration at which we determined the association rate At such concentrations the fraction of RNA found in a complex with CBP2 would be small.
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Dissociation rates were determined by incubating renatured RNA and CBP2 for 1 hour to ensure complete binding and subsequently trapping any protein that dissociated by addition of heparin to a final concentration of 20 to 500 μg/ml Fraction RNA bound was analyzed by filter binding (4). Dissociation rates were independent of heparin concentration. End-points were obtained from reactions in which the heparin was added before CBP2
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23
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4243193346
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note
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-1) = 30 pM.
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We thank P. Bevilacqua for discussion. Supported by a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (K.M.W.) and by the Howard Hughes Medical Institute (T.R.C.).
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