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Chapter 9 : Mechanism of Transcriptional Activation by NtrC
In the case of nitrogen regulatory protein C (NtrC), both nucleotide hydrolysis and transcriptional activation depend on phosphorylation of an aspartate residue in the N-terminal receiver domain of the protein (also called its regulatory domain). In this chapter, the authors review the evidence that the NtrC protein from enteric bacteria, which is a dimer in solution, must form an appropriate oligomer to hydrolyze nucleotide and activate transcription. Because phosphorylation of the N-terminal receiver domain of NtrC is known to increase oligomerization, effects of phosphorylation on NtrC function may be a consequence of effects on oligomerization. Recent evidence from the laboratory indicates that oligomerization determinants of NtrC are located in its central activation domain. Studies of NtrC function have been facilitated by the use of two sorts of tools: mutant forms of NtrC and derivatives of the glnA enhancer. NtrC must be phosphorylated in its receiver domain to hydrolyze ATP and activate transcription. Phosphorylation stimulates the oligomerization of NtrC, and oligomerization is, in turn, required for ATP hydrolysis and transcriptional activation. Because the phosphorylated receiver domain of NtrC functions positively, it is presumably needed for appropriate oligomerization: removing this domain by proteolysis or genetic engineering does not substitute for phosphorylation. However, the unphosphorylated protein is essentially incapable of ATP hydrolysis or transcriptional activation, even when bound to the enhancer.
Domain structure of NtrC (reviewed by Kustu et al., 1991; D. Weiss et al., 1992; Morett and Segovia, 1993). NtrC is composed of three domains: an amino-terminal receiver domain (regulatory domain), a central activation domain, and a carboxy-terminal DNA binding and dimerization domain. The N-terminal domain (∼120 residues) contains the site of phosphorylation, aspartate 54 (D54). It is joined to the remainder of the protein by a flexible glutamine-rich linker (Q-linker). The central activation domain (∼240 residues) contains the “phosphate loop” (ATP binding motif), which is known to bind the β-phosphate in several nucleotide binding proteins. Oligomerization determinants lie in this domain (Flashner et al., submitted; see text). The C-terminal region of NtrC (∼90 residues) contains a helix-turn-helix DNA binding motif and the major dimerization determinants of the protein. Forms of NtrC lacking the N-terminal domain (ΔN-NtrC and ΔN-NtrCS160F) are missing residues 1 to 133 and begin within the Q-linker. Other activators of σ54-holoenzyme carry a domain homologous to the central domain of NtrC. Some, but not all, of these activators carry an N-terminal receiver domain.
Transcriptional activation by NtrC. σ54-holoenzyme by itself (Eσ54) binds to a promoter to form closed complexes, in which the DNA remains double-stranded. NtrC binds to a nearby enhancer, which is composed of two binding sites for dimers (A). Conserved promoter sequences recognized by the polymerase lie at —12 and —24 with respect to the start site of transcription. For the glnA gene of Salmonella typhimurium, the binding sites that constitute the enhancer lie at -108 and -140. As is the case for eukaryotic transcriptional enhancers, this enhancer functions efficiently at a distance of kilobases from the promoter and downstream as well as upstream of it (Reitzer and Magasanik, 1986; Reitzer et al., 1989). A phosphorylated oligomer of NtrC contacts σ54-holoenzyme by means of DNA loop formation (B). To activate transcription, NtrC catalyzes isomerization of closed complexes between polymerase and the promoter to open complexes, in which there is a region of localized strand denaturation around the transcriptional start site (C). To catalyze open complex formation, NtrC must hydrolyze ATP. In open complexes, the conformation of polymerase is changed such that its DNase I footprint is elongated and extends downstream of the transcriptional start site.
glnA enhancer and variants of it. (A) glnA regulatory region. (B) Sequence of the two dyad-symmetrical sites that compose the glnA enhancer. Regions of (imperfect) dyad symmetry are underlined. To produce the “strong enhancer” (C), a single site with increased dyad symmetry was substituted for sites 1 and 2. To produce the “weak enhancer” (D), the dyad symmetry of site 2 in the glnA enhancer was destroyed. (E) The “single symmetrical site” has perfect dyad symmetry. (Modified from Fig. 1 of Porter et al., 1993.)
Occupancy of the weak enhancer and the single symmetrical binding site by NtrCS160F (A) and transcriptional activation from each (B). Occupancy was assessed by DNase I footprinting at 2 nM DNA, the same concentration used for measuring transcriptional activation (the rate of open complex formation). Conditions used to determine occupancy and transcriptional activation were the same. Templates were linear and carried either the weak enhancer or the single symmetrical site at a distance of ∼400 bp upstream of the glnA promoter. (Modified from Fig. 5C and D in Porter et al., 1993.)
Synergy of activation by phosphorylated wild-type NtrC and phosphorylated NtrC3ala on a template carrying a single binding site for NtrC. Stimulation of open complex formation by the NtrC3ala protein (concentrations indicated on the x axis) was assessed in the presence of 5 nM wild-type NtrC (closed circles) or in the absence of wild-type NtrC (open circles). NtrB was present at 100 nM to phosphorylate the NtrC proteins. The supercoiled template, which carries a single symmetrical binding site for NtrC ∼400 bp upstream of the glnA promoter, was at 20 nM. (From Fig. 6B in Porter et al., 1993.)
Effect of spacing between NtrC binding sites on transcriptional activation (the rate of open complex formation). The effect of the spacing between the two binding sites that constitute the strong enhancer on the rate of open complex formation at the glnA promoter was assessed in a single-cycle transcription assay. Rates are expressed in finol transcript/5 min. The NtrCD54E,S160F constitutive mutant protein, which has higher activity than the NtrCS160F form (Klose et al., 1993), was titrated on templates carrying two strong binding sites separated by three turns of the DNA helix (center-to-center) (Fig. 3C; closed circles), four turns of the helix (closed triangles), or 2.5 turns (open circles). It was also titrated on a template carrying a single strong NtrC binding site (open squares). Templates were fragments of ∼700 bp carrying sites ∼400 bp upstream of the glnA promoter (Porter, 1993; Porter et al., 1993). Specifically, they were (i) the 703-bp KpnI-PstI fragment from pJES534 (three turns); (ii) the 712-bp KpnI-PstI fragment from pJES587 (four turns); (iii) the 698-bp KpnI-PstI fragment from pJES639 (2.5 turns); and (iv) the 635-bp KpnI-PstI fragment from pJES520 (single site). Under the conditions used, transcription from the glnA promoter on a template lacking NtrC binding sites was undetectable in the range of concentrations of NtrCD54E,S160F shown.
Effect of the strong enhancer, a single strong site, or DNA lacking NtrC binding sites on ATPase activity of NtrC. Sites were carried on double-stranded oligonucleotides of 69 bp. The oligonucleotides carried the strong enhancer or derivatives of it in which one or both sites were replaced with random DNA. ATPase assays were performed in acetate buffer, as described (Weiss et al., 1991), and results are expressed in nmol Pi released (A) or pmol Pi released (B and C) during the time indicated. NtrC proteins (NtrCS160F for A and B and wild-type [WT] NtrC for C) were present at the concentrations indicated and were incubated with oligonucleotides for 5 to 10 min at 37°C before the addition of ATP. For the experiments in B and C, NtrB was present at 100 nM to phosphorylate the NtrC proteins, and they were phosphorylated for 10 min at 37°C in the presence of unlabeled ATP before the labeled nucleotide was added.