Fig 1.
Nucleic acid scaffolds used in association with E. coli core RNA polymerase to produce well-defined elongation complexes.
Each scaffold contained an RNA primer annealed to a 30 nt DNA template strand that is associated with a 30 nt fully complementary non-template strand. (A) The scaffold containing the 9mer RNA was used in the stopped-flow kinetic studies. (B) The scaffold containing the 10mer RNA was used in the quenched flow-thin layer chromatography (QF-TLC) studies. The base pair given in red indicates the alteration in the DNA sequence that was made to limit the incorporation of nucleotides to only AMP followed by CMP.
Fig 2.
Stopped-flow kinetic results for pyrophosphate release during one (A) and two rounds (B) of the NAC.
The reactions were monitored by using a coupled enzyme assay as outlined in the experimental section. In each case, a control in the absence of the EC corresponding to the background was subtracted from the reaction in the presence of the EC and nucleotide(s). The concentration of the EC after mixing was 0.2 μM; 25°C in each case. (A) UTP final concentration after mixing was 50 μM; the pyrophosphate release curve corresponds to the average of three runs; and the solid line through the data points corresponds to the fit to Scheme 1. (B) UTP and ATP concentrations after mixing were both 50 μM; the pyrophosphate release curve corresponds to the average of two runs; and the solid line through the data points corresponds to the fit to Scheme 4. (C) Residual plot for the fit of the data from a single round of the NAC. (D) Residual plot for the fit of the data from two rounds of the NAC.
Table 1.
Values and limits of rate constants for E. coli RNA core polymerase during one round of the NAC based on the model given in Scheme 1 for UTP binding to the pre-translocated state as monitored in stopped flow kinetic studies.
Fig 3.
Analysis of the stopped flow kinetic data set for a single round of the NAC assuming that nucleotide incorporation is the rate limiting step in the cycle.
(A) The data set shown is the same as that given in Fig 2A for a single round of the NAC at 25°C. The solid black line through the data points corresponds to the fit to Scheme 1 when the value of the rate constant for nucleotide incorporation was set equal to 10 s-1. (B) Residual plot for the fit of the data.
Table 2.
Values and limits of rate constants for E. coli RNA polymerase during one round of the NAC based on the model given in Scheme 1 for UTP binding to the pre-translocated state as monitored in stopped flow kinetic studies.
Nucleotide incorporation is assumed to be the rate limiting step.
Fig 4.
Stopped-flow kinetic results for pyrophosphate release during one round of the NAC with the EC undergoing translocation prior to NTP binding.
(A) The data set shown is the same as that given in Fig 2A for a single round of NAC at 25°C. The reaction was monitored by using a coupled enzyme assay as outlined in the experimental section. The solid black line through the data points corresponds to the fit to Scheme 2. (B) Residual plot for the fit of the data.
Table 3.
Values and limits of rate constants for E. coli RNA polymerase during one round of the NAC based on the model given in Scheme 2 showing NTP binding to the post-translocated state as monitored in stopped flow kinetic studies.
Fig 5.
Analysis of the pyrophosphate release curve over the region of 0.08 to 0.7 sec from the study reporting rapid pyrophosphate release from the EC during a single round of the NAC following CMP incorporation.
(A) The concentration of the EC after mixing was 0.2 μM and the CTP final concentration after mixing was 200 μM. The line through the data points corresponds to the fit to Scheme 3. (B) Residual plot for the fit of the data. (Inset) Variation of the pyrophosphate release curve over the range of 1 to 7 seconds along with a linear fit to the data.
Table 4.
Values and limits of rate constants for E. coli RNA core polymerase during one round of the NAC based on the model given in Scheme 3 for the incorporation of CMP and the release of pyrophosphate.
Table 5.
Values and limits of rate constants for E. coli RNA polymerase during two rounds of the NAC based on the model given in Scheme 4 as monitored in stopped flow kinetic studies.
Table 6.
Values and limits of rate constants for E. coli RNA polymerase in the presence of the cognate first NTP (UTP) and noncognate second NTPs (CTP and GTP), respectively, for incorporation during one round of the NAC based on the model given in Scheme 1 for NTP binding to the pre-translocated state as monitored in stopped flow kinetic studies.
Table 7.
Values of rate constants for E. coli core RNA polymerase during one round of the NAC in the presence of [γ-32p]ATP based on the model given in Scheme 5 for ATP binding to the pre-translocated state as monitored in QF-TLC studies.
Fig 6.
Efficacies of using phosphate and pyrophosphate mops together (A) or a phosphate mop alone (B) to eliminate background fluorescence due to the corresponding contaminants present in the nucleotide samples.
The final concentration of UTP after mixing in the stopped-flow apparatus in each case was 50 μM. The protocol for conducting this study is the same as that given in the methods section except that the elongation complex is not present.
Fig 7.
Stopped-flow kinetic results for pyrophosphate release in the presence of UTP and CTP (A) as well as UTP and GTP (B).
NTP concentrations were each 50 μM with EC concentration of 0.2 μM after mixing at 25°C. The appropriate control in the absence of the EC corresponding to the background was subtracted in each case. The pyrophosphate release curve for UTP and CTP corresponds to the average of four runs and the curve for UTP and GTP corresponds to the average of two runs. The solid black lines through the data points correspond to the fits to Scheme 1.
Fig 8.
Control for QF-TLC studies illustrating the purity of [γ-32P]ATP and that there is no time-dependent variation in the intensity of the band corresponding to [γ-32P]PPi.
(A) In the autoradiogram, each lane corresponds to a quench at the indicated time. The concentration of [γ-32P]ATP [60 μCi/pmol] after mixing was 50 μM. A duplicate is given in S1 Raw images along with a larger version of the image in Fig 8A. (B) Plot of the intensity of the [32P]PPi contaminant spot over time. The data correspond to the average of two independent experiments. For analysis, each data set was normalized by dividing the intensity of each spot by the average of the intensities for the seven time points. The normalized values of the two sets of data were then averaged and the standard deviation of each time point was determined.
Fig 9.
QF-TLC analyses of pyrophosphate release during one and two rounds of the NAC.
The autoradiograms correspond to the time-dependent release of [32P]PPi in the presence of (A) 0.5 μM EC and 50 μM [γ-32P]ATP [60 μCi/pmol] or (B) 0.5 μM EC with 50 μM [γ-32P]ATP [60 μCi/pmol] and 50 μM CTP, respectively, after mixing at 25°C. (C) Averages for each data set in C were obtained as indicated in Methods. The non-zero y-intercept in each case is due to contaminating [32P]PPi in the reaction mixtures. (●) Plot of the time dependent release of [32P]PPi in the presence of 0.5 μM EC and 50 μM [γ-32P]ATP [60 μCi/pmol] after mixing. The data correspond to the average of four independent experiments. The line through the data points was generated by fitting the data to the model in Scheme 5. (●) Plot of the time dependent release of [32P]PPi in the presence of 0.5 μM EC, 50 μM [γ-32P]ATP [60 μCi/pmol] and 50 μM CTP after mixing at 25°C. The data correspond to the average of four independent experiments. The line through the data points was generated by fitting the data to the model in Scheme 6. (E) Residual plot for the fit of the data for one round of the NAC. (D) Residual plot for the fit of the data for two rounds of the NAC.
Table 8.
Values of rate constants for E. coli core RNA polymerase during two rounds of the NAC in the presence of [γ-32P]ATP and CTP as monitored by QF-TLC.
Fig 10.
QF-TLC results for pyrophosphate release in the presence of [γ-32P]ATP and UTP as well as [γ-32P]ATP and GTP, respectively, during one round of the NAC.
The autoradiograms correspond to the time-dependent release of [32P]PPi in the presence of (A) 0.5 μM EC, 50 μM [γ-32P]ATP [60 μCi/pmol] and 50 μM UTP at 25°C and (B) 0.5 μM EC with 50 μM [γ-32P]ATP [60 μCi/pmol] and 50 μM GTP, respectively, after mixing at 25°C. Averages for each data set in C and E were obtained as indicated in Methods. The non-zero y-intercept in each case is due to contaminating [32P]PPi in the reaction mixtures. (C) Plot of the time dependent release of [32P]PPi in the presence of 0.5 μM EC, 50 μM UTP and 50 μM [γ-32P]ATP [60 μCi/pmol] after mixing. The data correspond to the average of three independent experiments. The line through the data points was generated by fitting the data to the model in Scheme 5. Plot of time dependent release of [32P]PPi in the presence of 0.5 μM EC, 50 μM GTP and 50 μM [γ-32P]ATP [60 μCi/pmol] after mixing at 25°C (E). The data correspond to the average of two independent experiments. The line through the data points was generated by fitting the data to the model in Scheme 5.
Table 9.
Values of rate constants for E. coli core RNA polymerase during one round of the NAC in the presence of [γ-32P]ATP and the noncognate nucleotides as monitored by QF-TLC.
Fig 11.
An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription.
Template DNA strand is shown in black, RNA strand in red, incoming NTP along with PPi in green and Mg2+ in yellow. BH is the bridge helix which separates the active site from the downstream DNA, TL is the trigger loop and FL is the fork loop. The active site corresponds to i + 1 and the position one nucleotide downstream from the active site corresponds to i + 2.