An Autoinhibited State in the Structure of Thermotoga maritima NusG

Drögemüller, Johanna; Stegmann, C.; Mandal, A.; Steiner, Thomas; Burmann, Björn M.; Gottesman, Max E.; Wöhrl, Birgitta M.; Rösch, Paul; Wahl, Markus C.; Schweimer, Kristian

doi:10.1016/j.str.2012.12.015

Cited by 16 publications

(29 citation statements)

References 40 publications

(64 reference statements)

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“…The plasmid pETM11-tmNusGΔ R275A,R279A coding for tmNusGΔ R275A,R279A (11) was used as template for site-directed mutagenesis to generate tmNusGΔ R275A,R279A,F336A with the following primers: 5′-gta aac gta act ata gcc gga cgt gaa act cc-3′, 5′- gg agt ttc acg tcc ggc tat agt tac gtt tac -3′. Point mutations were introduced according to the QuikChange kit (Agilent Technologies, Santa Clara, CA, USA) and confirmed by sequencing (GATC Biotech, Köln, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…The proteins were produced and purified as described (11). The protocol for tmNusGΔ was also used for all its variants.…”

Section: Methodsmentioning

confidence: 99%

“…The NTD and CTD of NusG from the hyperthermophilic bacterium Thermotoga maritima ( T. maritima , tmNusG) tightly interact with each other, in striking contrast to the corresponding domains of ecNusG (11,12). As the binding sites for RNAP, NusE and Rho are masked in this closed state of tmNusG, the respective interactions are supposed to be repressed (11).…”

Section: Introductionmentioning

confidence: 99%

“…As the binding sites for RNAP, NusE and Rho are masked in this closed state of tmNusG, the respective interactions are supposed to be repressed (11). Compared with ecNusG, tmNusG comprises an additional domain, DII, that is inserted into tmNusG-NTD (11). tmNusG-DII is composed of two subdomains and interacts neither with tmNusG-NTD nor with tmNusG-CTD.…”

Section: Introductionmentioning

confidence: 99%

“…tmNusG-DII is composed of two subdomains and interacts neither with tmNusG-NTD nor with tmNusG-CTD. An insertion is also present in NusG from Aquifex aeolicus (aaNusG) which, however, is significantly smaller than tmNusG-DII and shows no sequential or structural homology to tmNusG-DII (11,13–14). Although non-sequence specific binding to nucleic acids has been demonstrated for the additional domain in both tmNusG and aaNusG, the functions of these domains remain unclear (13,15).…”

Section: Introductionmentioning

confidence: 99%

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Thermotoga maritimaNusG: domain interaction mediates autoinhibition and thermostability

et al. 2016

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NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures.

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Section: Methodsmentioning

confidence: 99%

“…The proteins were produced and purified as described (11). The protocol for tmNusGΔ was also used for all its variants.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Thermotoga maritimaNusG: domain interaction mediates autoinhibition and thermostability

et al. 2016

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Ubiquitous transcription factors display structural plasticity and diverse functions

NandyMazumdar

Artsimovitch

2015

BioEssays

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Numerous accessory factors modulate RNA polymerase response to regulatory signals and cellular cues and establish communications with co-transcriptional RNA processing. Transcription regulators are astonishingly diverse, with similar mechanisms arising via convergent evolution. NusG/Spt5 elongation factors comprise the only universally conserved and ancient family of regulators. They bind to the conserved clamp helices domain of RNA polymerase, which also interacts with non-homologous initiation factors in all domains of life, and reach across the DNA channel to form processivity clamps that enable uninterrupted RNA chain synthesis. In addition to this ubiquitous function, NusG homologs exert diverse, and sometimes opposite, effects on gene expression by competing with each other and other regulators for binding to the clamp helices and by recruiting auxiliary factors that facilitate termination, antitermination, splicing, translation, etc. This surprisingly diverse range of activities and the underlying unprecedented structural changes make studies of these “transformer” proteins both challenging and rewarding.

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