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

Abstract

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 ...

TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

“…The model of the complex is consistent with cross-linking of both DksA and TraR to Q933 in the trigger loop (16) (Fig. 1C) and positions the N-terminal region of TraR to make a close approach to the active site region of RNAP, like DksA (14,16,17).…”

“…We describe a model for TraR action in which TraR mimics the combined effects of ppGpp and DksA by using the residues in RNAP that help form ppGpp site 2. TraR adds to the growing repertoire of known factors-including DksA, GreA, GreB, and Rnk (15)(16)(17)(18)(19)(20)-that target the β′ rim helices of RNAP to modulate transcriptional output. Members of the TraR class of…”

“…These include: the β′ secondary channel rim, including residue E677, which interacts with the DksA globular domain (14,17,28); the RNAP active site region at the base of the secondary channel and the trigger loop, which interact with the DksA coiled-coil tip (16,17,29) (Fig. 1C); and the sequence insertion 1 (SI1) subdomain in the nearby β-subunit, which binds to the C-terminal helix of DksA (17).…”

The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein

“…In addition, there are examples where fruitful alternatives other than NMR, X-ray crystallography, or electron microscopy (EM) have been used to obtain accurate structure–function information about composite multidomain proteins. For example, crosslinkable derivatives of both nucleotides and amino acids have been successfully employed in studies elucidating binary and ternary complexes of bacterial DNA-dependent RNA polymerase (RNAP) with DNA [66] and elongation factors [67]. Depending on the chemical nature of the reagent (crosslinkable moiety, length of the spacer), it might be possible to study both intra- and intermolecular interactions within the p53 tetramer, alone or in complex with DNA.…”

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

“…It is formed by the C and N termini of DksA and harbors a CXXCX 17 CXXC zinc finger motif (13). In addition, recent work in E. coli identified the RNAP ␤ subunit sequence in-sertion 1 as a binding site for the DksA C-terminal helix (16). Structural as well as amino acid substitution analyses of DksA proteins from Pseudomonas aeruginosa, Rhodobacter sphaeroides, and E. coli indicate that the conserved amino acid motif DXXDXA at the tip of the CC domain is critical for DksA function as a transcriptional regulator (17)(18)(19).…”