Fluorescence-based assay to measure the real-time kinetics of nucleotide incorporation during transcription elongation

Abstract

Understanding the mechanism and fidelity of transcription by the RNA polymerase (RNAP) requires measurement of the dissociation constant (K(d)) of correct and incorrect NTPs and their incorporation rate constants (k(pol)). Currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. Here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. The fluorescent adenine analogue 2-aminopurine was incorporated at various single positions in the template or the nontemplate strand of the promoter-free elongation substrate. On addition of the correct NTP to the T7 RNAP-DNA, 2-aminopurine fluorescence increased rapidly and exponentially with a rate constant similar to the RNA extension rate obtained from the radiometric assay. The fluorescence stopped-flow assay, therefore, provides a high-throughput way to measure the kinetic parameters of RNA synthesis. Using this assay, we report the k(pol) and K(d) of all four correct NTP additions by T7 RNAP, which showed a range of values of 145-190 s(-1) and 28-124 μM, respectively. The fluorescent elongation substrates were used to determine the misincorporation kinetics as well, which showed that T7 RNAP discriminates against incorrect NTP both at the nucleotide binding and incorporation steps. The fluorescence-based assay should be generally applicable to all DNA-dependent RNAPs, as they use similar elongation substrates. It can be used to elucidate the mechanism, fidelity, and sequence dependency of transcription and is a rapid means to screen for inhibitors of RNAPs for therapeutic purposes.

Fig.1. 2-Aminopurine modified elongation substrates and their fluorescence properties with and without T7 RNAP and NTP

(A) A:T base pair compared to 2AP:T and 2AP:U base pairs. (B) The elongation substrate showing the positions where a single 2AP was incorporated (denoted as P). (C) 2AP fluorescence was collected over 360-400nm with excitation at 310 nm and the intensity was normalized to that of individual 2AP-contained free elongation substrate. Relative fluorescence are shown for substrates alone with 2AP at T_n+1 or T_n (200nM C/2AP-T_n+1 or 2AP/2AP-T_n, with T7 RNAP (400nM), and with RNAP and the next correct NTP (400 μM). (D) Relative fluorescence of substrate with 2AP at NT_n+1 (200nM A/2AP-NT_n+1), with T7 RNAP, and with RNAP and UTP, UTP + ATP, or the incorrect CTP (400 μM each). The 3’-dC-SC1A contains a 3’-deoxyCMP (3’-dC) base at the 3’-terminus of the RNA primer.

Fig. 2. Elongation substrate with 2AP in the template strand monitors the kinetics of GMP incorporation

(A) The general scheme of the stopped-flow experimental set up consisted of loading a mixture of T7 RNAP (400 nM) and 2AP-labeled elongation substrate (200nM) in one syringe and NTP in the second syringe. The two solutions were rapidly mixed to initiate the RNA synthesis reaction. The mixed sample was excited with 310 nm light and fluorescence was measured at >360 nm. (B) The elongation substrate (C/2AP-T_n+1) contained a 2AP (P) in the template strand at n+1 position. The C in the template at position n will base pair with GTP. Upon GTP addition, the RNAP moves into the new position where 2AP occupies the n position. (C) Representative time trace at 50 μM GTP shows a single exponential increase (smooth curve) in 2AP fluorescence with k_obs = 77 ± 4 s^-1. (D) GTP concentration dependence of the observed rate constant (k_obs) fit to a hyperbolic function (Eq. 2) with a maximum rate constant k_pol = 190 ± 10 s^-1 and ground-state dissociation constant K_d = 70 ± 10 μM. Error bars encompass high and low values from duplicate measurements. (E) C/2AP-T_n+1 substrate with 3’-dC-RNA primer did not show a fluorescence change upon mixing with GTP (200 μM).

Fig. 3. Elongation substrate with 2AP in the nontemplate strand monitors the kinetics of CMP incorporation

(A) The G/2AP-NT_n elongation substrate contains a 2AP in the NT at the n-position and correctly base pairs with CTP. (B) A representative time trace of 2AP fluorescence change at 40 μM CTP (200nM G/2AP-NT_n) and 400 nM T7 RNAP). The experimental data fit well to a single exponential equation (Eq.1) (solid line) with k_obs= 136 ± 24 s^-1. (D) The k_obs increased with increasing [CTP] and fit to a hyperbola (Eq. 2) with a maximum rate constant k_pol = 209 ± 27 s^-1 and K_d = 28 ± 13 μM. Error bars indicate the high and low values from duplicate measurements.

Fig. 4. Stopped-flow kinetics of multiple UMP incorporation

(A) The A/2AP-NT_n+1 contains 2AP in the NT strand at n+1 position and correctly base pairs with UTP. (B) Representative time traces of 2AP fluorescence changes upon adding UTP (1, 5, 50, and 200 μM, final concentrations) shows multiple kinetic phases. (C) The k_obs-1 of the fast phase increases with [UTP] in a hyperbolic manner with k_pol-1 = 192 ± 20 s^-1 and K_d-1 = 98 ± 22 μM. Error bars indicate standard deviation from multiple measurements. (D) UTP concentration dependence of the slower phase, k_obs-2 shows a linear dependence with slope = 0.002 μM^-1s^-1 representing k_pol-2/K_d-2 ratio (D) UTP concentration dependence of the fast (open triangle) and slow phase (filled circle) amplitudes of 2AP fluorescence changes fit to the hyperbolic equation (Eq.2) with K_d,net = 0.3 μM for correct UMP:dA incorporation and K_d,net2 = 32 μM for UMP:dA misincorporation .

Fig. 5. Fluorescence measurements supporting the slow phase of UMP:dA misincorporation

(A) Measurement of 3’-dUMP incorporation in A/2AP-NT_n+1. (B) A representative time course of 2AP fluorescence change at 50 μM 3’-dUTP fits to a single exponential k_obs = 39 s^-1. (C) 3’-dUTP concentration dependence of k_obs fits to a hyperbola with k_pol = 235 ± 28 s^-1 and K_d = 185 ± 64 μM. Error bars encompass the high and low values from duplicate measurements. (D) Measurement of successive UMP and AMP incorporation in A/2AP-NT_n+1. (E) Representative time traces show multiple 2AP fluorescence changes with 300μM UTP alone (black trace), UTP + 30μM ATP (red trace), and UTP + 300 μM ATP (green trace).

Fig. 6. UMP incorporation during RNA synthesis measured by the radiometric assay

(A) The polyacrylamide sequencing gels show 10-mer RNA elongated to 11-13 mer RNAs with increasing reaction times. Lanes 0-12 represent reaction times from 0, 2, 4, 6, 8, 10, 30, 50, 100, 500 msec, and 1, 5, 10 s at 10 μM (right side) or 100 μM (left side) UTP. (B) The kinetics of 11-13 mer RNA formation at UTP concentrations of 10μM (filled circles) and 100 μM (filled triangles) were fit to a single exponential equation (Eq.1) with k_obs = 24 s^-1 and 113 s^-1, respectively. (C) UTP concentration dependence of k_obs for correct UMP incorporation fit to the hyperbolic function (Eq. 2) with k_pol = 251 ± 23 s^-1 and K_d = 133 ± 20 μM. (D) UTP concentration dependence of the k_obs for rU:dA misincorporation fits to a line with slope = 0.001 μM^-1.s^-1. (E) UTP concentration dependence of the amplitudes of correct (filled circle) and incorrect (open circle) UMP incorporation. A net dissociation constant for UMP misincorporation (K_d,net-mis= 263 μM) was derived from the hyperbolic fitting.

Fig. 7. Stopped-flow fluorescence measurements of G:dT misincorporation

(A) Addition of GTP to T/2AP-T results in rG:dT misincorporation. (B) Representative time traces of correct and incorrect NTP addition. The 2AP fluorescence increases upon adding the incorrect GTP (200 μM) with k_obs = 0.6 s^-1 and with the correct ATP (200 μM) with k_obs = 100 s^-1. (B) GTP concentration dependence of the misincorporation reaction rate constant fit to a hyperbola (Eq. 2) with k_pol = 5.2 ± 0.6 s^-1 and K_d = 1816 ± 483μM.