Transcription by σ54-RNA Polymerase Is Controlled by Activators That Bind Far from the Promoter
The sequence of σ54 is distinctly different from that of all the σ70-like factors. Transcription of genes by RNA polymerases containing σ54 is regulated solely by activators whose binding sites in DNA, referred to as enhancers, are generally located 80–160 bp upstream from the transcription start site. Even when enhancers are moved more than a kilobase away from a start site, σ54-activators can activate transcription.
The best-characterized σ54-activator—the NtrC protein (nitrogen regulatory protein C)—stimulates transcription of the glnA gene. The glnA gene encodes the enzyme glutamine synthetase, which synthesizes the amino acid glutamine, the central molecule of nitrogen metabolism, from glutamic acid and ammonia. The σ54-RNA polymerase binds to the glnA promoter but does not melt the DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein. NtrC, in turn, is regulated by a protein kinase called NtrB. In response to low levels of glutamine, NtrB phosphorylates dimeric NtrC, which then binds to an enhancer upstream of the glnA promoter. Enhancer-bound phosphorylated NtrC then stimulates the σ54-polymerase bound at the promoter to separate the DNA strands and initiate transcription.
Electron microscopy studies have shown that phosphorylated NtrC bound at enhancers and σ54-polymerase bound at the promoter interact directly, forming a loop in the DNA between the binding sites (Figure 9-5). As discussed later in this chapter, this activation mechanism resembles the predominant mechanism of transcriptional activation in eukaryotes.
EXPERIMENTAL FIGURE 9-5 DNA looping permits interaction of bound NtrC and σ54-RNA polymerase. (a) Drawing (left) and electron micrograph (right) of DNA restriction fragment with phosphorylated NtrC dimers bound to the enhancer region near one end and σ54-RNA polymerase bound to the glnA promoter near the other end. (b) Drawing (left) and electron micrograph (right) of the same fragment preparation, showing NtrC dimers and σ54-RNA polymerase bound to each other, with the intervening DNA forming a loop between them. See W. Su et al., 1990, Proc. Natl. Acad. Sci. USA 87:5504.
[Micrographs courtesy Harrison Echols and Carol Gross.]
NtrC has ATPase activity, and ATP hydrolysis is required for activation of bound σ54-RNA polymerase by phosphorylated NtrC. Mutants with an NtrC that is defective in ATP hydrolysis are invariably defective in stimulating the σ54-RNA polymerase to melt the DNA strands at the transcription start site. It is postulated that ATP hydrolysis supplies the energy required for melting the DNA strands. In contrast, the σ70-polymerase does not require ATP hydrolysis to separate the strands at a start site.