Press Release

RIKEN 

Regulation through the secondary channel - structural framework for ppGpp-DksA synergism during transcription

- Crystal structure and molecular mechanism of transcription factor DksA -

6 August 2004

  Bacterial transcription is regulated by the alarmone ppGpp, which binds near the catalytic site of RNA polymerase (RNAP) and modulates its activity. We show that the DksA protein is a crucial component of ppGpp-dependent regulation. The 2.0Åresolution structure of Escherichia coli DksA revealed a globular domain and a coiled-coil with two highly conserved Asp residues at its tip, which is reminiscent of the transcript cleavage factor, GreA. This structural similarity suggests that the DksA coiled-coil protrudes into the RNAP substrate entry (secondary) channel to coordinate a ppGpp-bound Mg²⁺ ion with the Asp residues, thereby stabilizing the ppGpp-RNAP complex. Biochemical analyses demonstrated that DksA affects transcript elongation, albeit differently from GreA, augments the ppGpp effects on initiation, and binds directly to RNAP, positioning the Asp residues near the active site. Substitution of these residues eliminated the synergy between DksA and ppGpp. Thus the secondary channel has emerged as a common regulatory entrance for transcription factors.

1. Background
  Stringent control, a complex series of regulatory events in bacterial cell starved for amino acids, is triggered by the elevated concentrations of guanosine-tetraphosphate (ppGpp, also known as "magic spot"). Complexed with RNAP, this nucleotide selectively regulates the transcription of genes involved in amino acid metabolism. On one hand, ppGpp inhibits the transcription of rRNA and tRNA genes, while on the other, it stimulates the expression of proteins required for amino acid biosynthesis and transport. Thus, the overall effect of ppGpp action is to increase the amino acid pools in the cell.
  An intriguing and unresolved discrepancy exists between the small, albeit reproducible, ppGpp effects in highly purified in vitro systems and the dramatic range of regulation observed in vivo. This apparent discrepancy could be due to a requirement for cellular factor(s) that modulate ppGpp activity in vivo - in fact, the existence of such an auxiliary factor was proposed nearly 30 years ago. Recently, the DksA protein was shown to greatly amplify the inhibition of rRNA promoters by ppGpp in vitro, and thus DksA may play the role of the missing in vivo modulator of the ppGpp activities.

2. Results
  In this work, we show the surprising result that the DksA protein, although lacking any sequence similarity, closely resembles the structure of another well known transcription factor, GreA. Both contain a long α-helical coiled-coil domain with invariant acidic residues at the tip. It was proposed recently that upon binding to RNAP, the coiled-coil of GreA protrudes deeply into the substrate entry (secondary) channel towards the RNAP active site, where its invariant acidic residues coordinate the catalytic Mg²⁺ ion.
  We have recently determined the RNAP/ppGpp complex structure (Cell, 2004, 117, 299-310), which revealed that ppGpp binds in the RNAP secondary channel in close vicinity to the RNAP active site. The structure also identified two Mg²⁺ ions bound to each of the di-phosphates of ppGpp. Whereas one of the ppGpp-bound Mg²⁺ ions is buried within the protein and is anchored well by the protein residues, the second Mg²⁺ ion is accessible from the outside through the RNAP secondary channel and is loosely bound by only the ppGpp phosphates.
  Given the previously demonstrated modulation of ppGpp activity by DksA and the structural similarity with GreA, the DksA structure implies a molecular mechanism in which DksA, like GreA, binds to RNAP, with its coiled coil protruding through the secondary channel towards the ppGpp binding site, and stabilizes the RNAP/ppGpp complex through coordination of the loosely ppGpp-bound Mg²⁺ by the invariant acidic residues. To verify the proposed mechanism we have carried out a limited set of the focused biochemical experiments, which showed that DksA indeed directly binds to RNAP and positions the tip of its coiled coil near the RNAP active site, and that the invariant acidic residues are crucial for the DksA function, because mutations of these residues resulted in the loss of the DksA effect on the ppGpp activity.

3. Perspectives
  This work illustrates a scenario in which the mechanism of action of a regulatory factor could be deduced from a cursory analysis of its three dimensional architecture, given that it resembles another, well-studied protein. The close match between the DksA and GreA structures allowed us to conduct a rapid and detailed biochemical analysis of the functional mechanism of DksA using a very limited set of genetic data as the starting point.
  In combination with the existing data, our results provide two major important implications that extend beyond the functional mechanism of DksA itself, towards the more general, fundamental principles of transcription regulation. First, the RNAP secondary channel emerges as a major binding site for a rapidly expanding group of transcription regulators that require access to the RNAP catalytic center to "tune" it for various regulatory activities. We may expect that other, as yet unknown, transcription factors could also modulate the RNAP activity by using their extended functional segments to reach into the secondary channel towards their targets.
  Another important implication is that the binding of a nucleic acid backbone to RNAP could be achieved not only through the direct hydrogen bonding of basic residues to the nucleotide phosphates, but also through Mg²⁺-mediated interactions, to which the protein would donate acidic ligands. One apparent candidate for such a mode of stabilization is an initiating nucleotide, which is likely to be loosely bound in the open complex in the absence of the RNA transcript. Coordination of the Mg²⁺ ion bound to the phosphates of the initiating nucleotide by the protein acidic residues might enhance its binding affinity and thereby facilitate transcription initiation.

Supplementary Explanations

X-ray diffraction
About 1Åof X-ray wavelength is almost the same as the distance between atoms in a substance, e.g. a protein, and is diffracted by a crystal its substances regularly arranged components. By analyzing the diffracted X-ray intensities, the a molecular structure in the crystal can be understood.

Angstrom Å
This is a unit of length, and 1 angstrom is 1x10^-10m(=0.1nm). For three-dimensional structure analyses of proteins, this unit is used to representing the resolution of the analyzed structures. Smaller angstrom values represent, higher resolution and thus a more precise three-dimensional structure.

Coiled-coil domain
A domain is functionally and structurally a primordial unit of a protein, and a "coiled-coil" domain is one with two alpha helices coiled around each other.

Helix
One of the higher-order protein structures. A helix structure is a spiral structure with 3.6 amino acid residues per turn, and the rise per residue is 1.5Å along the axis. Secondary Channel Pathway for a substance that is influenced by an enzyme interaction.
"in vivo" and "in vitro" "In vivo" is a term used to describe what reactions occurring within living cells or organisms. "In vitro" is a term used to distinguish reactions occurring outside of organisms from those within organisms.

Upper panel. Model of the RNAP/DksA/ppGpp complex (overview)

Lower panel. Close up view of the ppGpp binding site of the RNAP/DksA/ppGpp complex.