Purification and properties of stringent factor.
The stringent factor from Escherichia coli is the product of the relA locus. It is the enzyme that catalyzes the synthesis of pppGpp and ppGpp eliciting a pyrophosphate transfer from ATP to the 3'--OH of GTP (or GDP). This protein is responsible for the synthesis of pppGpp and ppGpp in stringent strains in response to an amino acid starvation. In vitro it catalyzes the synthesis of these guanosine compounds in either a ribosome-dependent reaction that requires a particular conformation of the ribosome i.e. the presence of an uncharged tRNA recognizing a codon in the acceptor (A) site of the ribosome or in a ribosome-independent reaction at temperatures under 30 degrees in the presence of only buffer, salts, and substrates. Here we report the purification of the stringent factor to near homogeneity. It is a monomeric protein with a molecular weight of 75,000. The properties of the ribosome-independent reaction are studied and it is shown that the presence of certain acidic proteins, such as the 50 S ribosomal proteins L7 and L12 or casein, or 20% methanol or both stimulates the reaction by creating an environment that together with the low temperature further stabilizes the stringent factor.
D000255:Adenosine Triphosphate; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000096:biosynthesis; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D010756:Phosphoric Acids; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology
D006150:Guanine Nucleotides; D006882:Hydroxyapatites; D010756:Phosphoric Acids; D000255:Adenosine Triphosphate; D010770:Phosphotransferases