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ABSTRACT Tuberculosis is caused by the bacteriumMycobacterium tuberculosis(Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species,Mycobacterium smegmatis(Msm), perform the essential function of RNA synthesis, we performed a series ofin vitrotranscription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacteriumMycobacterium tuberculosis(Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie,et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.