Protein synthesis by cell-free extracts of Proteus vulgaris

Abstract

BACKLER, K. L., and W. E. INNISS. 1971. Protein synthesis by cell-free extracts of Proteus vulgaris.Can. J. Microbial. 17: 1545-1551. A cell-free protein-synthesizing system from Proteus vrtlgaris was developed and characterized. Initially, a very low activity was found to occur; it was shown to be due to a deficiency in the endogenous transfer ribonucleic acid (tRNA) content of the cell extracts. When tRNA was added to the polyuridylic acid (poly U) directed system, incorporation of 14C-phenylalanine into polypeptide readily occurred. This incorporation was dependent on ribosomes and supernatant fraction and the optimun~ concentrations of the various components of the system were found to be as follows: 120 pglml tRNA, 30 m M magnesium, 0.5 m M spermidine, 60 m M potassium, 50 m M adenosine triphosphate (ATP), 5 m M guanosine triphosphate (GTP), 0.03 m M cytosine triphosphate (CTP) and uridine triphosphate (UTP), 5 m M phosphoenolpyruvate (PEP), 50 p g l n~l poly U, and 30 m M 2-mercaptoethanol. Un systen~e de synthese proteinique dtpourvu &elements cellulaires a ete realise et caracterise i~ partir d'une souche de Proterrs vrtlgaris. Au depart, I'activite du systeme s'est averee tres faible en raison d'une deficience dans le contenu en acide ribonucleique de transfert (ARN-t) endogene de I'extrait cellulaire. L'addition d'ARN-t au systeme dependant de l'acide polyuridylique (poly U) facilite la prolnpte incorporation de la phenylalanine 14C dans le polypeptide. Cette illcorporation etait sous la dependance des ribosomes et de la fraction surnageante. Les concentrations opti~num des divers composants du systeme ont Cte Ctablies colnme suit: ARN-t, 120 pglml; magnesium, 30 mM; spermidine, 0.5 mM; potassiunl, 60 mM; adenosine triphosphate (ATP), 50 mM; guanosine triphosphate (GTP), 5 mM; cytosine triphosphate (CTP) et uridine triphosphate (UTP), 0.03 mM; phosphoenolpyruvate (PEP), 5 mM; poly U, 50 pg/ml et 2-nlercaptoethanol, 30 n1M.