Bacterial RNA synthesis: back to the limelight

AbstractTranscription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

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Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gkz1221 ◽

2020 ◽

Vol 48 (4) ◽

pp. 2144-2155 ◽

Cited By ~ 3

Author(s):

Yeonoh Shin ◽

Mark Hedglin ◽

Katsuhiko S Murakami

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Initiation Complex ◽

X Ray ◽

The Core ◽

Core Enzyme ◽

Bacterial Rna ◽

Reiterative Transcription ◽

Transcript Initiation ◽

Template Dna

Abstract Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak–trigger–freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.

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Transcription inhibition by the depsipeptide antibiotic salinamide A

eLife ◽

10.7554/elife.02451 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 52

Author(s):

David Degen ◽

Yu Feng ◽

Yu Zhang ◽

Katherine Y Ebright ◽

Yon W Ebright ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Transcription Initiation ◽

Rna Synthesis ◽

Antibacterial Drug ◽

Transcription Inhibition ◽

Bacterial Rna ◽

Cellular Target ◽

Nucleotide Addition ◽

And Function

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.

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