Xia et al. 10.1073/pnas.0506181102. |
Fig. 7. Initial signal intensity measured in fluorescence upconversion (Upper) and transient absorption (Lower) collected in a short time window for free 2-aminopurine (2Ap), free boxB RNA, 11-mer peptide/boxB complex, or 22-mer peptide/boxB (WT) complex.
Fig. 8. Effects of substitution of Glu-13 by proline on the dynamics measured in both transient absorption (Top) and fluorescence upconversion experiments (Middle) and on transcription antitermination activity level in vivo (Bottom) for Lys-14–Gln-15 (WT), Lys-14–Arg-15, and Glu-14–Arg-15 complexes.
Supporting Experimental Procedures
Femtosecond Laser Spectroscopy.
The experimental set-up used in the femtosecond fluorescence up-conversion and transient absorption experiments has been published previously (see, e.g., ref. 1). Briefly, femtosecond pulses (<100 fs; 800 nm; »1.5 mJ) were generated from a Ti:sapphire laser (Spectra-Physics). The pulse was split equally to pump two optical parametric amplifiers (OPAs). The signal output from one OPA was quadrupled to generate a pump pulse at 325 nm. In fluorescence up-conversion experiments, the fundamental 800 nm from the other OPA was used as the probe pulse; in the transient absorption experiments, the signal output from the OPA was doubled to 600 nm to generate the probe pulse. In all of the experiments, the temperature of the sample quartz cell (5-mm path length) was controlled at 20°C or at room temperature, and the samples were stirred. All of the data were fitted to a multiexponential decay convoluted by a Gaussian response function as described in ref. 1.Femtosecond Transient Absorption.
Transient absorption procedures are described in refs. 1 and 2. The 2Ap containing RNA was excited by a pump pulse (325 nm; 20–50 nJ). A second pulse at 600 nm, delayed in time, was used to measure the absorbance of the 2Ap excited state (2Ap*) in the complexes, or also as free in solution. The polarization of the pump and probe pulses was set at the magic angle (54.7°) to avoid complications from orientational motions.Femtosecond Fluorescence Up-Conversion.
Femtosecond fluorescence up-conversion procedures are described in refs. 1 and 3. The 2Ap was excited by a pump pulse (325 nm; 200 nJ), and care was taken to ensure that the low power regime was valid. The emission was collected by a pair of parabolic focus mirrors and mixed with the fundamental (800 nm) in the beta barium borate (BBO) crystal. The up-converted signal (257 nm) was detected by a photomultiplier after passing through a double-grating monochromator. The pump beam polarization was set at the magic angle with respect to fluorescence polarization set by the BBO crystal.Steady-State Fluorescence.
Steady-state fluorescence measurements were conducted on a Shimadzu Spectrofluorophotometer at 20°C with excitation/emission wavelengths at 310/370 nm as described in ref. 4. Peptides were titrated iteratively into a constantly stirred solution of 2Ap-labeled RNA hairpin (20–200 nM RNA). Binding buffer contained 20 mM Tris•OAc, with a variable concentration of KOAc (15–500 mM) at pH 7.5. Binding constants were obtained for a one-step binding mechanism by using nonlinear least-squares regression with the aid of a computer program, DYNAFIT (5).Imino Proton NMR Spectroscopy.
Imino proton NMR spectra were collected on a Varian INOVA 600 MHz at 25°C in a buffer of 50 mM NaCl, 10 mM phosphate, and 0.5 mM EDTA (pH 6). Complex formation was monitored by inspecting the RNA imino protons. The concentrations of the complexes were 150–300 mM.Circular Dichroism (CD).
CD spectra were collected on an 62 DS CD (Aviv Associates, Lakewood, NJ) spectrophotometer in 10 mM potassium phosphate buffer (pH 7) at 20°C. Spectra for the peptide portion in the complex were calculated by subtracting spectra for free RNA and excess peptide from that of the complex. Helical content was calculated as described in ref. 4.1. Fiebig, T., Wan, C. & Zewail, A. H. (2002) Chem. Phys. Chem. 3, 781–788.
2. Wan, C., Fiebig, T., Schiemann, O., Barton, J. K. & Zewail, A. H. (2000) Proc. Natl. Acad. Sci. USA 97, 14052–14055.
3. Pal, S. K., Peon, J. & Zewail, A. H. (2002) Proc. Natl. Acad. Sci. USA 99, 1763–1768.
4. Austin, R. J., Xia, T., Ren, J., Takahashi, T. T. & Roberts, R. W. (2002) J. Am. Chem. Soc. 124, 10966–10967.
5. Kuzmic, P. (1996) Anal. Biochem. 237, 260–273.