CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase

Abstract

The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.

Figure 1.

Schematic of bacterial transcription initiation and the structure of the Mtb RNAP-σ^A holoenzyme. (A) A kinetic model of initiation where the promoter DNA (P) containing the template strand (cyan) and non-template strand (purple) with a Cy3-fluorescent label positioned at the +2 position (yellow star) undergoes a fluorescence increase when RNAP (R, grey circle) unwinds the DNA during the isomerization from RP_c to RP_o. NTP addition results in scrunched RP_itc intermediates, where the open DNA bubble is increased in size. Transcription complexes can undergo either abortive cycling (red box) between RP_itc and RP_o states or productive synthesis (green box). Promoter escape leads to a fluorescence quenching when the DNA strands re-anneal in the vicinity of the Cy3-probe. (B) Mtb RNAP-σ^A holoenzyme rrnAP3 open promoter complex with CarD and RbpA (PDB: 6EDT) (30). Cryo-EM structure containing the Mtb rrnAP3 promoter (–60 to +30) with a fully open DNA bubble. Cy3-labeling position for use in stopped-flow experiments shown in yellow. Black box indicates a 90^o rotation of the region shown in (C) where the predicted interactions of σ^A, CarD and RbpA with the open DNA bubble are shown. The Mg²⁺ ion indicates the position of the RNAP active site.

Figure 2.

Real-time traces of RP_o formation as indicated by the increase in fluorescence relative to rrnAP3 DNA alone. Final concentrations are 50 nM Eco holo or Mtb holo with 1 μM CarD, 2 μM RbpA, or 1 μM CarD and 2 μM RbpA mixed with 1 nM rrnAP3 DNA. An average of five independent shots per condition is plotted as fold-change over DNA alone.

Figure 3.

Dissociation from Mtb RP_o as a function of heparin and salmon-sperm DNA concentration. 50 nM Mtb holo pre-incubated with 1 nM rrnAP3 DNA titrated against various competitor concentrations (concentrations listed are following equal volume mixing). Data is plotted as percent of signal remaining at a given time. Averages of at least three shots are depicted. Black lines indicate fits to a sum of three exponentials for (A) heparin and a sum of two exponentials for (B) salmon-sperm DNA. Insets depict competitor titrations on a linear time-scale.

Figure 4.

Transient peak signal present at high NTP concentrations is due to initial transcribing complexes. (A) Comparison of dissociation and promoter escape for Mtb. Mtb holo RP_o mixed with 25 μg/ml salmon-sperm DNA, 50 μM NTPs, and 1000 μM NTPs (concentrations following equal volume mixing). Inset shows the percent increase above the starting fluorescence signal plotted from 0.001 to 5 s. (B) NTP-subset experiments for Mtb. Mtb holo RP_o mixed with salmon-sperm DNA only (RP_o, red) or with salmon-sperm DNA and initiating nucleotide (GTP, RP_itc1, blue), nucleotides sufficient for formation of a 4-mer (G/UTP, RP_itc4, yellow), nucleotides sufficient for formation of a 5-mer (G/UTP and 3’-O-methyl-CTP, RP_itc5, brown), nucleotides sufficient for formation of a 9-mer (G/U/CTP, RP_itc9, green) or all four nucleotides to allow escape (RP_e, purple). NTP additions yielded a final concentration of 1000 μM for each NTP following equal volume mixing. Inset depicts peak signal plotted from 0.001 to 100 s with the initially transcribing sequence of the non-template strand listed above.

Figure 5.

Eco displays faster initial nucleotide incorporation but slower promoter escape kinetics than Mtb. 50 nM Mtb (green) or Eco holo (orange) pre-incubated with 1 nM rrnAP3 DNA mixed with 25 μg/ml salmon-sperm DNA and 1 mM NTPs. Inset shows the percent increase above the starting fluorescence signal plotted between 0.001 and 5 s.

Figure 6.

CarD and RbpA effects on Mtb promoter escape kinetics and inactive fraction. (A) NTP titrations for Mtb holo. NTP conditions range from 0 to 2 mM after equal volume mixing. Inset presents the transient peak signal as a percentage increase above the initial fluorescence value plotted from 0.001 to 5 s. Increasing the NTP concentration decreases the time-to-peak, as indicated by the dotted black line and arrow. (B) Overlay of escape traces conducted at 1 mM NTPs for Mtb holo ± factors. Inset depicts data on a linear time-scale. (C) Quantification of t_1/2^escape values and (D) inactive fraction as calculated from the data in (B). Error bars represent standard errors of the mean.

Figure 7.

NTP dependence of the the peak signal. (A) Time-to-peak and (B) corresponding peak amplitudes as a function of NTP concentration for Mtb holo with and with-out factors. Error bars represent standard errors of the mean. (C) Time-to-peak simulations where the net exit rate is fixed while increasing the net entry rate into RP_itc and (D) the net entry rate is fixed while decreasing the net exit rate out-of RP_itc (see Supplementary Figures S16 and S17). Here, the darkening trend represents larger changes from the starting value and increases in the peak amplitude.

Figure 8.

CarD and RbpA effects on RP_itc intermediates. (A) Overlay of peak signal obtained at 1 mM NTPs for Mtb holo ± factors plotted as a percentage increase above the initial fluorescence value. (B) Simulations of factor dependent effects on peak signal. The combined RP_o and RP_itc signals are plotted as a percent increase above the initial fluorescence signal. Factor dependent effects on k_itc and k_escape were adjusted according to the experimentally determined fold changes in the time-to-peak relative to Mtb holo alone. CarD (blue) is modeled by a 0.4-fold decrease in the net exit rate, RbpA (red) is modeled by a 0.9-fold decrease in the net entry rate. In the case of both factors, simulations adjusting both the net entry and exit rates as in the individual factor simulations were unable to reproduce the experimentally observed results in the time-to-peak and amplitude (light purple). Simulating a 0.75-fold decrease in both the net entry and exit rates, yielded a similar amplitude and time-to-peak as observed experimentally in the presence of both factors (dark purple).

Figure 9.

Differential regulation in flux is dependent upon the basal rates of initiation. (A) Three-state model for calculations of steady-state flux where CarD and RbpA either increase (green arrow) or decrease (red line) specific rate constants. Factor-dependent fold-changes in transcript flux show both activation (green) and repression (red) as a function of the basal opening and escape rates for (B) RbpA and (C) CarD. Promoters that are rate-limited at escape (i.e. fast k_open, slow k_escape) are predicted to be repressed, whereas promoters that are rate-limited at opening (i.e. slow k_open, fast k_escape) are predicted to be activated.