Structural Basis for Transcription Regulation by Alarmone ppGpp

Abstract

Guanosine-tetraphosphate (ppGpp) is a major regulator of stringent control, an adaptive response of bacteria to amino acid starvation. The 2.7 A resolution structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in complex with ppGpp reveals that ppGpp binds to the same site near the active center in both independent RNAP molecules in the crystal but in strikingly distinct orientations. Binding is symmetrical with respect to the two diphosphates of ppGpp and is relaxed with respect to the orientation of the nucleotide base. Different modes of ppGpp binding are coupled with asymmetry of the active site configurations. The results suggest that base pairing of ppGpp with cytosines in the nontemplate DNA strand might be an essential component of transcription control by ppGpp. We present experimental evidence highlighting the importance of base-specific contacts between ppGpp and specific cytosine residues during both transcription initiation and elongation.

Still Looking for the Magic Spot: The Crystallographically Defined Binding Site for ppGpp on RNA Polymerase Is Unlikely to Be Responsible for rRNA Transcription Regulation

“…20 We suggest that neither the wild-type nor the mutant E. coli promoter complexes were actually affected by ppGpp in the previous work (Fig. 4B in ref.…”

“…3B and 3C), the one mutant RNAP examined previously (in ref. 20 ; see Discussion). Saturating concentrations of ppGpp also decreased the half-lives of the complexes formed by the seven other mutant RNAPs that were tested, and the effects were at least as great as with the wild-type and β′ N458S RNAPs (column 3, Table 2; for additional plots, see Supplementary Fig.…”

“…6B and data not shown), in contrast to the conclusion reached previously (in ref. 20 ; see Discussion). Promoter complexes formed by RNAP from another Thermus species, T. aquaticus, were also unaffected by ppGpp (data not shown).…”

“…1A). 20 Two different orientations of ppGpp were present in the two complexes in the cocrystal's asymmetric unit, with ppGpp "flipped" in one complex relative to the other. The 5′-diphosphate was located closer (proximal) to the active site Mg 2+ in one orientation, and the 3′-diphosphate was closer to the active site Mg 2+ in the other orientation.…”

“…19,20,21,22,23 It was claimed that T. thermophilus RNAP responded directly to ppGpp in vitro, and that an N458S substitution in the β′ subunit of E. coli RNAP, corresponding to a residue contacting ppGpp in the T. thermophilus RNAP cocrystal, decreased the ability of the promoter complex to respond to ppGpp. 20 Finally, it was proposed that the guanine base in ppGpp pairs directly to cytosines on the nontemplate DNA strand in the open complex, just upstream of the transcription start site.…”