Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

Hubin, Elizabeth A.; Lilić, Mirjana; Darst, Seth A.; Campbell, Elizabeth A.

doi:10.1038/ncomms16072

Cited by 61 publications

(62 citation statements)

References 67 publications

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“…We found that ~25% of putative ECF σ promoters contained at least one UP-element subsite. UP-element subsites were preferentially located at the proximal, distal, and far-distal subsites ( Figure 4C), and the frequency of UP-elements varied by bacterial phyla, consistent with previous reports (Table 2; Hubin et al 2017).…”

Section: Predicted Ecf σ Promoters Are Similar To Experimentally Charsupporting

confidence: 91%

Rewiring the specificity of extra-cytoplasmic function sigma factors

Todor

Osadnik

Campbell

et al. 2020

Preprint

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Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here, we address these challenges by solving the DNA-specificity code of extra-cytoplasmic function sigma factors (ECF σ s), a major family of bacterial regulators, and determining their regulons.We generated an aligned collection of ECF σ s and their promoters by leveraging the autoregulatory nature of ECF σ s as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid -nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic foot-printing pipeline based on precomputed orthologs to predict the direct targets of ~67% of ECF σ s. This global survey indicated that ECF σ s play varied roles: some are global regulators controlling many genes throughout the genome that are important under many conditions, while others are local regulators, controlling few closely linked genes in response to specific stimuli. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

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Section: Predicted Ecf σ Promoters Are Similar To Experimentally Charsupporting

confidence: 91%

Rewiring the specificity of extra-cytoplasmic function sigma factors

Todor

Osadnik

Campbell

et al. 2020

Preprint

Self Cite

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“…Holoenzyme structures complexed with RNA (PDB ID 5VT0 (62)) (A and C) and promoter DNA (PDB ID 5VI5 (84))(B and D) are centered on region 4.2 of σ 70 and shown without (A and B) or with (C and D) the nucleic acids visible. The cryoEM structure with 6S RNA is E. coli holoenzyme and the crystal structure with DNA in an open complex is Mycobacterium smegmatis holoenzyme and thus there are small variations in structure due to sequence changes, although region 4.2 of σ 70 is very highly conserved.…”

Section: Figurementioning

confidence: 99%

6S RNA, a Global Regulator of Transcription

Wassarman

2018

Microbiol Spectr

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6S RNA is a small RNA regulator of RNA polymerase that is present broadly throughout the bacterial kingdom. Initial functional studies in Escherichia coli revealed that 6S RNA forms a complex with RNA polymerase resulting in regulation of transcription, and cells lacking 6S RNA have altered survival phenotypes. The last decade has focused on deepening the understanding of several aspects of 6S RNA activity including: 1. Addressing questions of how broadly conserved 6S RNAs are in diverse organisms through continued identification and initial characterization of divergent 6S RNAs; 2. The nature of the 6S RNA-RNA polymerase interaction through examination of variant proteins and mutant RNAs, crosslinking approaches, and ultimately a cryo-EM structure; 3. The physiological consequences of 6S RNA function through identification of the 6S RNA-regulon and promoter features that determine 6S RNA sensitivity; 4. The mechanism and cellular impact of 6S RNA-directed synthesis of pRNAs (i.e. pRNA synthesis). Much has been learned about this unusual RNA, its mechanism of action and how it is regulated; yet, much still remains to be investigated, especially regarding potential differences in behavior of 6S RNAs in diverse bacteria.

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“…However, the structural details between 1.1 domains from different organisms vary widely. Currently, three types of known structures of this domain exist: (i) the highly similar structures, as solved for B. subtilis and E. coli, consisting of three helices (HI to HIII) packed in antiparallel manner (8,10); (ii) the structure from T. maritima, where HI packs perpendicularly to HII and HIII (9); and (iii) the possibly disordered and flexible structure from mycobacteria with only the A N-helix discernible (11)(12)(13)(14).…”

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confidence: 99%

The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

et al. 2019

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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.

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Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

Cited by 61 publications

References 67 publications

Rewiring the specificity of extra-cytoplasmic function sigma factors

Rewiring the specificity of extra-cytoplasmic function sigma factors

6S RNA, a Global Regulator of Transcription

The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

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