The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

Abstract

Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

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

“…The major change between the States is the interplay between the refolding of the PCh-loop and the CO-domain position in the primary channel. In State III, solely the PCh– loop’s tight contact with the AS stabilizes a very open form of RNAP (Figure S8a,b,f), ~33 Å at the narrowest point of the primary channel (measured by the distance of the Cα atoms of β/Lys273 and β’/Lys123), comparable to the structures Core2 and Holo2 1 (32.2 and 33.6 Å, respectively). In State I, the PCh–loop’s interaction with β’ helix-turn-helix and three-stranded sheet, and the CO-domain insertion into the primary channel make the opening of the RNAP clamp (~35 Å; Figure S8a,b) slightly wider than the extremely open forms of the Lipiarmycin-(PDB ID 6FBV 23 ) and Fidaxomicin-locked (PDB ID 6C06 24 ) RNAPs (34.2 and 33.6 Å, respectively 1 ).…”

“…A smoothly functioning transcription machinery is essential for maintaining the physiologically relevant levels of gene products and adequate changes in transcription are necessary for cell survival when the environment changes. In bacteria, transcription is executed by a single enzyme, DNA-dependent RNA polymerase [RNAP; composition of the core enzyme: α 2 ββ’ω 1 ]. The RNAP core is capable of transcription elongation and termination but not initiation.…”

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation

“…This conclusion is supported by the cryo-EM structure of Mycobacterium smegmatis σ A -RNAP holoenzyme lacking electron density for the domain σ4. This indicates that σ fluctuates between different conformational states ( Kouba et al, 2019 ). The smFRET study on σ B - Mtb RNAP also explains why RbpA is essential for transcription initiation at the −10/−35 promoters and dispensable at the extended −10 promoters ( Hu et al, 2012 , 2014 ; Perumal et al, 2018 ).…”

Molecular Basis for RNA Polymerase-Dependent Transcription Complex Recycling By The Helicase-like Motor Protein HelD.

“…4c ε is shown as it is clear that RNAP isolated from B. subtilis is a heterogeneous mixture of core (α 2 ββ'ω) ± δ, ε, and HelD, in addition to multiple different σ factors 21 . Mycobacterium smegmatis 6F6W, and 170.1 × 110.1 × 127.8 Å; Thermus thermophilus 2O5I) [22][23][24] , although it appears to be more slender and elongated than the roughly globular E. coli, and shorter than the M. smegmatis and T. thermophilus enzymes due to the lack of insertion sequences.…”