Biomaterial Database

Evidence for a ppGpp-binding site on Escherichia coli RNA polymerase: proximity relationship with the rifampicin-binding domain. 1995

P S Reddy, and A Raghavan, and D Chatterji

Centre for Cellular and Molecular Biology, Hyderabad, India.

On amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of ppGpp with a fluorescent probe, 1-aminonapthalene-5-sulphonate (AmNS), at the terminal phosphates. AmNS-ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titration of the tryptophan emission of RNA polymerase by AmNS-ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS-ppGpp with RNA polymerase. Forster's distance measurement was carried out which placed AmNS-ppGpp 27 A away from the rifampicin-binding domain of RNA polymerase.

UI	MeSH Term	Description	Entries
D009282	Naphthalenesulfonates	A class of organic compounds that contains a naphthalene moiety linked to a sulfonic acid salt or ester.
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005456	Fluorescent Dyes	Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.	Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006153	Guanosine Diphosphate	A guanine nucleotide containing two phosphate groups esterified to the sugar moiety.	GDP,Guanosine 5'-Diphosphate,Guanosine 5'-Trihydrogen Diphosphate,5'-Diphosphate, Guanosine,5'-Trihydrogen Diphosphate, Guanosine,Diphosphate, Guanosine,Diphosphate, Guanosine 5'-Trihydrogen,Guanosine 5' Diphosphate,Guanosine 5' Trihydrogen Diphosphate
D006159	Guanosine Tetraphosphate	Guanosine 5'-diphosphate 2'(3')-diphosphate. A guanine nucleotide containing four phosphate groups. Two phosphate groups are esterified to the sugar moiety in the 5' position and the other two in the 2' or 3' position. This nucleotide serves as a messenger to turn off the synthesis of ribosomal RNA when amino acids are not available for protein synthesis. Synonym: magic spot I.	Alarmone ppGpp,Bacterial Magic Spot ppGpp,Guanosine 5'-(trihydrogen diphosphate), mono(trihydrogen diphosphate) (ester),Guanosine 5'-diphosphate 2'(3')-diphosphate,ppGpp,Guanosine 3'-Diphosphate 5'-Diphosphate,Guanosine 5'-Diphosphate 3'-Diphosphate,3'-Diphosphate 5'-Diphosphate, Guanosine,5'-Diphosphate 3'-Diphosphate, Guanosine,Guanosine 3' Diphosphate 5' Diphosphate,Guanosine 5' Diphosphate 3' Diphosphate,Tetraphosphate, Guanosine,ppGpp, Alarmone
D006160	Guanosine Triphosphate	Guanosine 5'-(tetrahydrogen triphosphate). A guanine nucleotide containing three phosphate groups esterified to the sugar moiety.	GTP,Triphosphate, Guanosine
D001426	Bacterial Proteins	Proteins found in any species of bacterium.	Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D001667	Binding, Competitive	The interaction of two or more substrates or ligands with the same binding site. The displacement of one by the other is used in quantitative and selective affinity measurements.	Competitive Binding