Determination of RNA polymerase binding surfaces of transcription factors by NMR spectroscopy

Abstract

In bacteria, RNA polymerase (RNAP), the central enzyme of transcription, is regulated by N-utilization substance (Nus) transcription factors. Several of these factors interact directly, and only transiently, with RNAP to modulate its function. As details of these interactions are largely unknown, we probed the RNAP binding surfaces of Escherichia coli (E. coli) Nus factors by nuclear magnetic resonance (NMR) spectroscopy. Perdeuterated factors with [1H,13C]-labeled methyl groups of Val, Leu, and Ile residues were titrated with protonated RNAP. After verification of this approach with the N-terminal domain (NTD) of NusG and RNAP we determined the RNAP binding site of NusE. It overlaps with the NusE interaction surface for the NusG C-terminal domain, indicating that RNAP and NusG compete for NusE and suggesting possible roles for the NusE:RNAP interaction, e.g. in antitermination and direct transcription:translation coupling. We solved the solution structure of NusA-NTD by NMR spectroscopy, identified its RNAP binding site with the same approach we used for NusG-NTD, and here present a detailed model of the NusA-NTD:RNAP:RNA complex.

Mechanistic insights into transcription coupled DNA repair

“…This exposes the UvrB homology domain, which is presumed to recruit UvrA to the damage location [45]. It has been speculated that one monomer of UvrA interacts with Mfd, whereas the other monomer binds UvrB [46] (Fig. 1B).…”

“…NusA contacts the RNA exit channel by binding to the β flap tip helix of the β flap region of RNAP [53][54][46]. NusA might compete with Mfd, which also interacts with the RNAP β subunit [27][45][42].…”

Isotope-labeling strategies for solution NMR studies of macromolecular assemblies

“…Survey of recent literature shows many great examples of how this labeling strategy can generate exciting insights in the structure-dynamics-function relationship of protein-protein, protein-DNA, and protein-small molecule complexes involved in protein folding [29][30][31], regulation of protein expression [32][33][34][35][36], protein signal-transduction [37,38] and protein secretion [39][40][41]. We highlight here the work from the Kalodimos lab on the interaction of the 50 kDa trigger factor (TF) chaperone with a 48 kDa unfolded substrate, alkaline phosphatase (PhoA) [42 ].…”

Transcription Regulation in Archaea

“…Critical, direct molecular interactions between Spt5 and RNAP have been identified in both Bacteria and Archaea (83,84,87,88,95,(97)(98)(99), and the conservation of RNAP and Spt5 infers that these same interactions are used in Eukarya. Briefly, a hydrophobic depression on the NGN domain interacts with the mobile clamp domain of RNAP, with additional interactions between the NGN domain and RNAP jaw domain likely fixing the location of the clamp domain in a closed configuration (11,98).…”

“…The archaeoeukaryotic stalk, absent from bacterial RNAP, is used by a host of archaeal and eukaryotic transcription factors to bind and regulate the activities of RNAP. Increasing evidence from biochemical, biophysical, and in vivo approaches indicate that transcription factor binding often stimulates intramolecular movements of RNAP that appear necessary for transitions between phases of the transcription cycle (2,4,26,88,97,117).…”