Brezovska, B. | bioRxiv Science

bioRxiv2021

NAD+ capping of RNA in Archaea and Mycobacteria

Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD+ capping might be universal in all domains of life has not been proven yet, as information on this RNA modification is missing for Archaea. Likewise, this RNA modification has not been studied in the clinically important Mycobacterium genus. Here, we demonstrate that NAD+ capping occurs in the archaeal and mycobacterial model organisms Methanosarcina barkeri and Mycobacterium smegmatis. Moreover, we identify the NAD+-capped transcripts in M. smegmatis, showing that this modification is more prevalent in stationary phase, and revealing that mycobacterial NAD+-capped transcripts include non-coding small RNAs, such as Ms1. Furthermore, we show that mycobacterial RNA polymerase incorporates NAD+ into RNA, and that the genes of NAD+-capped transcripts are preceded by promoter elements compatible with {sigma}A/{sigma}F dependent expression. Taken together, our findings demonstrate that NAD+ capping exists in the archaeal domain of life, suggesting that it is universal to all living organisms, and define the NAD+-capped RNA landscape in mycobacteria, providing a basis for its future exploration.

molecular biology↗

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗