Structural Insights into the Stringent Response

Abstract

The stringent response allows bacteria to survive conditions of amino acid starvation through regulation of transcription by derivatives of guanosine triphosphate or guanosine diphosphate collectively called alarmone or ppGpp [for guanosine 3′-diphosphate 5′-triphosphate or guanosine 3′,5′-bis(diphosphate)]. Alarmone interacts with RNA polymerase (RNAP) to control its activity in a promoter-selective mechanism. Two groups report structural analysis of proteins involved in this stringent response. Hogg et al . analyzed the structure of the enzyme Rel Seq , which is responsible for both the production and degradation of ppGpp, from the Streptococcus dysgalactiae subspecies e quisimilis . The structure indicated that the two active sites are 30 Å apart. Two conformations of the protein were observed and binding of an unusual ppGpp derivative, guanosine 5′-diphosphate-2′:3′-cyclic monophosphate (ppG2′:3′p), to the hydrolase site appeared to lock the protein into a hydrolase-on and synthetase-off conformation. In the synthetase-on structure, GDP binding to a catalytically competent synthetase site coincided with a nonproductive, unliganded state at the hydrolase site. The protein crystallized lacked the ribosome-binding portion, which is responsible for regulating the balance of the catalytic activities in response to the presence of ribosome with nonacylated tRNAs. However, the structure suggests an allosteric mechanism for deactivating synthetase activity in response to conformational changes induced by nucleotide binding to the hydrolase domain. A second paper by Artsimovitch et al. that explored the structure of the Thermus thermophilus RNAP homoenzyme with ppGpp provided insight into how ppGpp can stimulate transcription at some promoters and inhibit transcription at others. The crystal contained two RNAP molecules, which appeared to bind ppGpp in alternate orientations. The two RNAP molecules were designated RNAP1, in which ppGpp was bound such that the 5′ diphosphate of ppGpp was closest to the active site Asp residues, and RNAP2, in which the ppGpp bound such that the 3′ diphosphate of ppGpp was closest to the active site Asp residues. These different ppGpp binding orientations produce differences between RNAP1 and RNAP2 in their active site conformations and in the conformation of subunits that interacted with the DNA promoter in the open complex. The RNAP1 conformation may have increased catalytic activity due to changes in the affinity of the active site for Mg 2+ ions, whereas the activity of RNAP in the RNAP2 conformation may be inhibited because binding of the second Mg 2+ ion is unfavorable. In addition, the RNAP2 conformation may allow ppGpp to bind to cytosines in the nontemplate DNA strand, thereby destabilizing the DNA open complex and inhibiting transcription. Thus, these two orientations of ppGpp may produce conformations of RNAP that have different activities. The interaction with the nontemplate DNA may contribute to the promoter-selective effects of ppGpp. T. Hogg, U. Mechold, H. Malke, M. Cashel, R. Hilgenfeld, Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response. Cell 117 , 57-68 (2004). [Online Journal] I. Artsimovitch, V. Patlan, S.-i. Sekine, M. N. Vassylyeva, T. Hosaka, K. Ochi, S. Yokoyama, D. G. Vassylyev, Structural basis for transcription regulation by alarmone ppGpp. Cell 117 , 299-310 (2004). [Online Journal]