Activation of glnA transcription by nitrogen regulator I (NRI)-phosphate in Escherichia coli: evidence for a long-range physical interaction between NRI-phosphate and RNA polymerase.
Growth of cells of Escherichia coli in nitrogen-limited medium induces the formation of glutamine synthetase, product of the glnA gene, and of other proteins that facilitate the assimilation of nitrogen-containing compounds. Transcription from the glnAp2 promoter of the glnALG operon requires the phosphorylation of nitrogen regulator I (NRI) and, for optimal transcription, the binding of NRI-phosphate to two sites that can be over 1,000 base pairs from the binding site for RNA polymerase. In other procaryotic genes, placement of an activator-binding site further upstream from the start site of transcription diminishes expression. To determine how NRI-phosphate activates transcription and why NRI-dependent transcription differs from activation in other systems, we constructed recombinant plasmids with small alterations between the binding sites for NRI-phosphate and RNA polymerase and between the two high-affinity NRI-binding sites. We demonstrate that tightly bound NRI-phosphate activated transcription from either side of the DNA helix when at least 30 base pairs separated NRI-phosphate from RNA polymerase. In contrast, activation from a partial NRI-binding site was effective only from one side of the DNA. We also observed that glnA expression was optimal when the two high-affinity NRI-binding sites were on the same side of the DNA helix. We explain these results on the basis of a hypothesis that a contact between RNA polymerase and NRI-phosphate bound to an upstream site determines the rate of glnA transcription.
Reitzer
L J
LJ
Department of Molecular and Cell Biology, University of Texas, Dallas,Richardson 75083-0688.
Movsas
B
B
Magasanik
B
B
D001426:Bacterial Proteins; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D051922:PII Nitrogen Regulatory Proteins; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; D012321:DNA-Directed RNA Polymerases; D005974:Glutamate-Ammonia Ligase