D Chatterji1, N Fujita, A Ishihama. 1. Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan. dipan@ccmb.ap.nic.in
Abstract
BACKGROUND: Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure. RESULTS: Here, the azido-derivative of ppGpp was synthesized from azido-GDP and [gamma-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the beta-subunit of RNA polymerase. CONCLUSION: It was observed that both the N-terminal and C-terminal domains of the beta-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp.
BACKGROUND: Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure. RESULTS: Here, the azido-derivative of ppGpp was synthesized from azido-GDP and [gamma-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the beta-subunit of RNA polymerase. CONCLUSION: It was observed that both the N-terminal and C-terminal domains of the beta-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp.