STRUCTURAL AND FUNCTIONAL STUDIES OF SIGMA54-DEPENDENT TRANSCRIPTIONAL ACTIVATORS

Open Access
Author:
Chen, Baoyu
Graduate Program:
Integrative Biosciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 26, 2008
Committee Members:
  • B Tracy Nixon, Committee Chair
  • Arthur Mallay Lesk, Committee Member
  • Kathleen Postle, Committee Member
  • William O Hancock, Committee Member
Keywords:
  • small angle x-ray scattering
  • AAA+ ATPase
  • two-component signal transduction
  • enhancer binding protein
  • sigma54
  • SAXS
  • NtrC
  • transcription
Abstract:
How microbes quickly sense and respond to environmental changes is an intriguing and fundamental question. Amongst the many strategies used to regulate their adaptive behaviors, sigma54-dependent gene transcription often plays a crucial role. In this system, the sigma54 protein recruits bacterial RNA polymerase (RNAP) to sigma54-specific promoters to form a stable complex. To start transcription, this complex needs actions from a bacterial enhancer binding protein (EBP). When stimulated by specific signals, an EBP assembles into a homo-oligomeric ring via its AAA+ ATPase (ATPases associated with various cellular activities) domain. The assembled ATPase utilizes ATP hydrolysis to remodel the closed sigma54/RNAP/promoter complex and enable it to initiate transcription of the downstream genes. This thesis mainly used small- and wide-angle X-ray scattering (SAXS/WAXS), gel filtration chromatography, and assay of ATP hydrolysis to explore the functional and structural details of three important aspects of this transcription regulatory system: 1) how the assembly of the ATPase is controlled; 2) how the assembled ATPase hydrolyzes ATP; and 3) how the ATPase uses ATP hydrolysis to remodel the sigma54/RNAP complex. Based on the results, first the thesis proposed a novel positive mechanism for how the assembly of an EBP ATPase is regulated by the receiver domain of two-component signal transduction, a frequent regulatory motif used by EBPs. Previous studies of two EBPs, NtrC1 and DctD, defined a negative regulation mechanism, in which the receiver domain inhibited the assembly of the ATPase while phosphorylation of the receiver domain released the inhibition and produced a constitutively active ATPase ring. However, this model failed to explain the regulation of the closely related NtrC. This study generated a low-resolution structural model that showed the receiver domain of NtrC, instead of inhibiting ring assembly, when phosphorylated actually stabilized it by associating with a neighboring ATPase subunit. The results provided the first structural model of an activated full-length EBP, explained previous biochemical and genetics data and solved the 20-year-old puzzle of the regulation of NtrC. Second, the thesis characterized the AAA+ ATPase domain of another EBP, NtrC1, at distinct stages during an ATP hydrolysis cycle. Each hydrolysis stage of the ATPase was captured by different nucleotides or analogues at saturating concentrations [e.g. ATP, ATPS, AMPPNP and ADP beryllium fluoride (ADP-BeFx) for the ground state, ADP aluminum fluoride (ADP-AlFx) for the transition state, and ADP for the product state]. The SAXS-derived low-resolution structures of the NtrC1 ATPase in different states revealed major conformational changes of the ATPase through the hydrolysis cycle. This work also defined a previously overlooked ATP ground state of EBP AAA+ ATPase as an important functional state for coupling conformational changes to sigma54 binding. Transitions between these conformations, especially movement of the highly conserved GAFTGA loops that are located on the top of the central pore region of the ATPase ring, were harnessed to perform mechanical work on the polymerase to initiate transcription. In summary, work in this thesis defined a new regulation mechanism of the assembly of the EBP ATPase controlled by two-component signal transduction, unambiguously revealed large conformational changes during an EBP ATPase cycle, and revised the current view of the functional and structural roles of the ATP hydrolysis ground state of EBP ATPases.