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

Abstract

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.