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A cell-free protein biosynthesizing system prepared from Escherichia coli CF300 was found to synthesize E. coli tryptophan synthase alpha subunit in a time-dependent manner when programmed with pBN69 plasmid DNA. This plasmid contains the trp promoter from Serratia marcescens adjacent to the coding region of E. coli tryptophan synthase alpha protein [Nichols, B.P., & Yanofsky, C. (1983) Methods Enzymol. 101, 155-164]. The synthesized tryptophan synthase alpha subunit was found to be indistinguishable from authentic alpha subunit protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have the same specific activity for catalyzing the conversion of indole----L-tryptophan by tryptophan synthase beta 2 subunit, as well as the conversion of indole + glyceraldehyde 3-phosphate to indole-3-glycerol phosphate. In the absence of exogenously added phenylalanine, admixture of E. coli phenylalanyl-tRNAPhe to the protein biosynthesizing system stimulated the production of functional alpha protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNAVal. The ability of a misacylated tRNA to participate in alpha protein synthesis in this system was established by the use of E. coli phenylalanyl-tRNAVal in the absence of added valine. Protein biosynthesis proceeded normally and gave a product having the approximate molecular weight of tryptophan synthase alpha subunit; as expected, this polypeptide lacked catalytic activity.

Uridine 5'-phosphate (UMP) synthase is a multifunctional protein that contains the last two enzyme activities for the de novo biosynthesis of UMP, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate (OMP) decarboxylase (EC 4.1.1.23). The native enzyme from mouse Ehrlich ascites cells exists in at least three distinct aggregation/conformation states as measured by sedimentation in sucrose gradients: a 3.6S monomer, a 5.1S dimer, and a conformationally altered 5.6S dimer. It has previously been reported that a variety of ligands (of which the most effective is OMP) mediate the conversion of the 3.6S monomer to the two types of dimers. Initial velocity studies with the enzyme in the different native states show that all three forms of UMP synthase have phosphoribosyltransferase activity but that the OMP decarboxylase is either uniquely or at least predominantly associated with the 5.6S form. Activation of this enzyme activity by the substrate appears to be the result of both a dimerization and a conformation step.

Several pyroglutamylaminoacyl-tRNA's were prepared by T4 RNA ligase mediated condensation of synthetic pyroglutamylaminoacyl-pCpA's with tRNA's from which the last two nucleotides at the 3'-end had been removed. The derived pyroglutamylaminoacyl-tRNA's were incubated in the presence of calf liver pyroglutamate aminopeptidase, which effected their conversion to free aminoacyl-tRNA's. The lack of contaminating esterase activities in the pyroglutamate aminopeptidase was verified by direct assay for the presence of the aminoacyl moieties in the formed aminoacyl-tRNA's and by the use of the deblocked aminoacyl-tRNA's as acceptors in the peptidyltransferase reaction using an Escherichia coli ribosomal system. These findings provide the wherewithal for a detailed investigation of the substrate specificity of the peptidyltransferase center and for the elaboration of polypeptides containing modified amino acids at predetermined sites.

Uridine 5'-phosphate (UMP) synthase is multifunctional protein that contains the last two enzyme activities of the de novo pathway for UMP biosynthesis, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate (OMP) decarboxylase (EC 4.1.1.23). We have previously reported that UMP synthase from mouse Ehrlich ascites cells can exist in at least three distinct aggregation and/or conformation states, as measured by changes in sedimentation through sucrose gradients [Traut, T. W., & Jones, M. E. (1979) J. Biol. Chem, 254, 1143-1150]. The major sedimenting species were a 3.6S monomer, a 5.1S dimer, and a 5.6S species. The formation of the 5.1S dimer from the 3.6S monomer occurs in the presence of ligands that are competitive inhibitors at the OMP decarboxylase catalytic site. This paper presents evidence for a regulatory site, distinct from either of the two catalytic sites, which appears to mediate the conversion of the 5.1S dimer to the 5.6S form upon binding certain pyrimidine nucleotides (OMP, UMP, and 6-azaUMP). Since UMP synthase sediments predominantly as a dimer in the presence of substrates, regulation of the aggregation/conformation state of this multifunctional protein may be physiologically significant.