DNA template sequence control of bacterial RNA polymerase escape from the promoter

Heyduk, Ewa; Heyduk, Tomasz

doi:10.1093/nar/gky172

Cited by 31 publications

(49 citation statements)

References 58 publications

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“…Our flux-based model implies that the outcome of CarD's activity at a promoter depends on intrinsic kinetics of the transcription initiation complexes that form at a particular promoter. In E. coli, RP o stability and promoter escape rates are highly dependent on sequences in the PRR (27,30,36). In addition, the small alarmone ppGpp binds to RNAP in E. coli to elicit bidirectional transcriptional responses by altering initiation kinetics (37), and the outcome of ppGpp activity is correlated with certain promoter DNA sequences (24).…”

Section: Discussionmentioning

confidence: 99%

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

Zhu

Garner

Galburt

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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The ability to regulate gene expression through transcription initiation underlies the adaptability and survival of all bacteria. Recent work has revealed that the transcription machinery in many bacteria diverges from the paradigm that has been established in Escherichia coli. Mycobacterium tuberculosis (Mtb) encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent in E. coli but is required to form stable RNAP-promoter open complexes (RPo) and is essential for viability in Mtb. The stabilization of RPo by CarD has been proposed to result in activation of gene expression; however, CarD has only been examined on limited promoters that do not represent the typical promoter structure in Mtb. In this study, we investigate the outcome of CarD activity on gene expression from Mtb promoters genome-wide by performing RNA sequencing on a panel of mutants that differentially affect CarD’s ability to stabilize RPo. In all CarD mutants, the majority of Mtb protein encoding transcripts were differentially expressed, demonstrating that CarD had a global effect on gene expression. Contrary to the expected role of CarD as a transcriptional activator, mutation of CarD led to both up- and down-regulation of gene expression, suggesting that CarD can also act as a transcriptional repressor. Furthermore, we present evidence that stabilization of RPo by CarD could lead to transcriptional repression by inhibiting promoter escape, and the outcome of CarD activity is dependent on the intrinsic kinetic properties of a given promoter region. Collectively, our data support CarD’s genome-wide role of regulating diverse transcription outcomes.

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Section: Discussionmentioning

confidence: 99%

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

Zhu

Garner

Galburt

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…PIFE effects using Cy3 at positions near the TSS were previously used to study aspects of the kinetics of OC formation, initiation, and escape. [45][46][47] Cy3 PIFE kinetic measurements were also used to study the cooperative interaction between CarD and RbpA transcription factor in OC formation for Mycobacterium tuberculosis σ A RNAP holoenzyme. [48][49][50] Here we use FRET and PIFE in kinetic assays of OC formation to investigate large DNA…”

Section: Introductionmentioning

confidence: 99%

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation byE. coliRNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

Sreenivasan

Shkel

Chhabra

et al. 2020

Preprint

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FRET (fluorescence energy transfer) between farupstream (-100) and downstream (+14) cyanine dyes showed extensive bending/wrapping of λPR promoter DNA on E. coli RNA polymerase (RNAP) in closed and open complexes (CC, OC).Here we determine the kinetics and mechanism of DNA bending/wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye fluorescence enhancements (PIFE). FRET/PIFE kinetics exhibit two phases: rapidly-reversible steps forming a CC ensemble ({CC}c of four intermediates (initial (RPC), early (I1E), mid-(I1M), late (I1L)), followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET/PIFE together reveal large-scale bending/wrapping of upstream and downstream DNA as RPC advances to I1E, reducing -100/+14 distance to ~75Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper b'-clamp while downstream DNA contacts the top of the b-pincer in I1E. Converting I1E to I1M (~1s time-scale) reduces FRET efficiency with little change in -100/+14PIFE, interpreted as clampopening that moves far-upstream DNA (on b') away from downstream DNA (on b) to increase the -100/+14 distance by ~14Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp-closing to reduce the -100/+14 distance by ~21Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L converts to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream-binding, DNA-bending transcription activators are discussed.

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“…Several groups using plant MoClo assembly have reported differences in cassette expression and functionality depending on position and orientations (e.g. 92), which highlights a key synthetic biology crux -the performance of a system is not simply the sum of its components (93,94).…”

Section: Resultsmentioning

confidence: 99%

CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

Vasudevan

et al. 2018

Preprint

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A Golden Gate-based toolkit for engineering cyanobacteria 2 ABSTRACT Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and exchange of parts between different labs and biological systems. However, a standardised Modular Cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential in biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. We have generated a suite of parts and acceptor vectors for making i) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and ii) transient multigene expression and repression systems using known and novel replicative vectors. We have tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The system is publicly available and can be readily expanded to accommodate other standardised MoClo parts.

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DNA template sequence control of bacterial RNA polymerase escape from the promoter

Cited by 31 publications

References 58 publications

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation byE. coliRNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

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