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.

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation byE. coliRNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

“…Rate and equilibrium constants for RPC formation(Table 3) are similar to published values for other promoters at similar conditions 49,50. The second order rate constant k1 (~3.0 x 10 8 M -1 s -1 ) for RPC formation is about 5% of the diffusion-collision limit, most simply interpreted as that only about 5% of collisions of R with P result in RPC formation.…”

“…[45][46][47] Cy3 PIFE kinetic measurements were also used to study the cooperative interaction between CarD and RbpA transcription factor in OC formation for Mycobacterium tuberculosis σ A RNAP holoenzyme. [48][49][50] Here we use FRET and PIFE in kinetic assays of OC formation to investigate large DNA…”

The dormancy‐specific regulator, SutA, is intrinsically disordered and modulates transcription initiation in Pseudomonas aeruginosa

“…In contrast to well‐studied activators that drive dramatic increases in transcription for a small subset of genes during an active stress response, by binding DNA and recruiting RNAP, SutA binds directly to RNAP, likely altering kinetic parameters that describe its interactions with the DNA during the OC formation. Factors that act in this way, including DksA/ppGpp, CarD and RbpA as well as SutA, potentially affect initiation at every promoter in a positive or negative way, depending on the characteristics of the promoter (Galburt, ; Jensen et al , ). The stimulatory effects of these factors on transcription initiation are never very large: from less than 2‐fold to 8‐fold for DksA/ppGpp on amino acid biosynthesis and stress response gene promoters (Paul et al , ; Blankschien et al , ; Girard et al , ); less than 2‐fold for RbpA on an rrn promoter; and 3‐ to 4‐fold for RbpA plus CarD on an rrn promoter (Hubin et al , ).…”

Conditional down-regulation of GreA impacts expression of rRNA and transcription factors, affecting Mycobacterium smegmatis survival

“…CarD specifically activated transcription from rRNA promoters in M.tuberculosis 28 . The activation mechanism involved stabilization of open complex formation by increasing the isomerization rate and decreasing the rate of DNA closing 42,44 . The decrease in CarD expression upon GreA depletion thus appears to adversely affect the rRNA transcription.…”