X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Murakami, Katsuhiko

doi:10.1074/jbc.m112.430900

Cited by 184 publications

(241 citation statements)

References 61 publications

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“…The movement of the two domains toward each other by 5-10 Å and 10-15 Å, respectively, could easily bring β-SI1 close enough to interact with DksA. DksA binding could thus capture an alternative conformation to that found in the crystal structure of E. coli RNAP (23,(25)(26)(27)(28) and compete with any function of β-SI1 associated with this original conformation. Although we have clearly demonstrated that β-SI1 recruits DksA to RNAP, further efforts both to dissect this novel binding interface and to characterize the DksA-independent functions of β-SI1 will be critical for completing the picture of how the interaction between β-SI1 and DksA alters transcription.…”

Section: Discussionmentioning

confidence: 99%

“…We propose that DksA-D74 functions during initiation by neutralizing the positive charges of β-R678/ R1106 and altering the dense network of polar-electrostatic interactions in the immediate vicinity of the active center (23,26,28,29). This could alter the conformation of two neighboring mobile elements of β, fork loop-1 and fork loop-2, destabilizing the intermediate on the pathway to open complex formation.…”

Section: Discussionmentioning

confidence: 99%

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DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

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

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Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo-cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.transcription regulation | stringent response | protein cross-linking | molecular modeling | lineage-specific insertions S ensing and responding to nutritional status is one of the major challenges of microbial life. In Escherichia coli, the global regulatory response to amino acid starvation is orchestrated by the second messenger guanosine-3′,5′-bisdiphosphate (ppGpp), which is a widely conserved master regulator (1). Accumulation of ppGpp during amino acid scarcity triggers the stringent response, which down-regulates expression of rRNA and tRNA while increasing expression of amino acid biosynthetic enzymes. In E. coli, ppGpp works synergistically with the transcription factor DksA to initiate the stringent response (2, 3). Both ppGpp and DksA are critical for survival of stress and virulence in many pathogenic proteobacteria (4).DksA is a relatively small protein with a prominent N-terminal coiled-coil domain and a globular C-terminal domain consisting of a Zn 2+ -binding region and a C-terminal α-helix (3). It belongs to a class of regulators that bind directly to RNA polymerase (RNAP) without contacting DNA (5). DksA modulates RNAP activity by preventing formation of or destabilizing the intermediate complex (RPi) on the pathway to the open complex (RPo), which is competent for initiation. For promoters with intrinsically unstable open complexes, such as rRNA promoters, DksA binding leads to decreased transcription (2).DksA is a critical determinant of the stringent response and a model system for an important class of transcription regulators, making it essential to understand how DksA interacts with RNAP at the molecular level. High-resolution structural information of the DksA/RNAP interaction is currently unavailable. Current models agree that the coiled-coil domain of DksA inserts into the secondary channel of RNAP, the channel used by NTPs to access the active site; that the secondary channel rim helices of β' subunit are critical for DksA binding; and that residues at the tip of the coiled-coil of DksA are important for its activity. However, the precise placement of DksA is unknown. With the number of critical features that can be accessed through the secondary channel, even small changes in the model can ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

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

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“…To better understand the roles of the REC-DBD interface in transcription activation, we dock the E. coli RNAPH 18 to the PmrA-DNA complex structure on the basis of the crystal structure of the s 4 -b-flap tip helix chimer/PhoB-DBD/DNA ternary complex 29 (Supplementary Fig. 14).…”

Section: Discussionmentioning

confidence: 99%

“…The docking involved use of PyMOL 47 . The starting structures for docking were the PmrA-DNA complex structure (complex-1) and the E. coli RNAPH structure 18 (PDB-ID: 4IGC). The crystal structure of the s 4 -b-flap tip helix chimer/PhoB-DBD/DNA ternary complex 29 (PDB: 3T72) was used as a model to drive the structure-based alignment.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, b-galactosidase reporter assay of PmrA variants with altered interface residues concludes that the formation of a stable REC-DBD interface is not crucial for activating downstream gene transcription. From the model docking the PmrA-DNA complex structure with the Escherichia coli RNA polymerase s 70 holoenzyme (RNAPH) 18 , the REC-DBD interdomain dynamics and the DBD-DBD interface of PmrA may help PmrA search for the most suitable conformation for interacting with the RNA polymerase holoenzyme to activate downstream gene transcription. Our combined X-ray and NMR studies of the PmrA-DNA complex illustrate the significant differences between the crystal and solution states for multipledomain proteins in bacterial two-component signal transduction.…”

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confidence: 99%

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Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

Hsiao¹

2016

Acta Cryst Sect A

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Sigma Factors in Gene Expression

Chandrangsu

Helmann

2014

Encyclopedia of Life Sciences

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Sigma (σ) factors control the promoter selectivity of bacterial RNA polymerase (RNAP). On binding to RNAP, σ factors allow efficient promoter recognition and transcription initiation. Bacterial promoters are typically comprised of two hexameric deoxyribonucleic acid (DNA) sequences located approximately 10 and 35 bases upstream of the transcription start site (the −10 and −35 element, respectively). All bacteria contain a primary σ factor that is responsible for transcription of housekeeping genes necessary for growth and survival. In addition, many bacteria encode multiple alternative σ factors. The level and activity of the alternative σ factors are highly regulated and can vary depending on environmental or developmental signals. The synthesis of alternative σ factors allows the coordinated activation of discrete sets of genes through the recognition of distinct promoter sequences and thereby contributes to stress responses, motility, endospore formation and numerous other adaptive responses. Key Concepts: σ Factors are necessary for promoter recognition by RNA polymerase and efficient transcription initiation. Transcription initiation is a highly regulated, multistep process. σ Factors can be grouped into two families on the basis of their similarity to the housekeeping σ factor, σ 70 , or the nitrogen‐responsive σ factor, σ 54 . Members of the σ 70 family generally contain four conserved domains (regions 1–4). Most bacteria encode multiple alternative σ factors that direct transcription of genes in response to environmental cues and developmental transitions. Alternative σ 70 family proteins often lack region 1 and sometimes region 3 (minimally containing conserved regions 2 and 4). σ Factor activity can be regulated at many levels, such as through sequestration by anti‐σ factors or by protolytic activation.

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X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Cited by 184 publications

References 61 publications

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

Sigma Factors in Gene Expression

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