A Temperature-sensitive thy Mutant Blocked in the Synthesis of Thymidylate Synthetase

thyA101 is a temperature-sensitive, thymine-requiring mutant of Escherichia coli B. At 37° it has approximately 1 to 2% of the thymidylate synthetase activity of the wild type organism. At 25° the level is about 30% of the wild type culture. These studies provide evidence that at 37° thy-101 is unable to complete the synthesis of thymidylate synthetase at the ribosomal level. On shifting a culture of thy-101 from 37° to 25°, a rapid formation of thymidylate synthetase occurs. The amount of this enzyme per unit of cell mass increases in two phases, first rapidly and then more slowly. A control enzyme such as glutamic acid dehydrogenase shows no evidence of a biphasic synthesis. Neither the rapid nor slow phase of thymidylate synthetase synthesis occurs in the presence of chloramphenicol or 5-methyltryptophan. To establish that the enzyme molecule is partially completed at 37°, thy-101 was grown at 37° in medium containing 90% deuterium oxide. The culture was then shifted to a water-containing medium at 25°. After a short period at 25°, an extract of the culture was prepared and subjected to separation by a CsCl centrifugal gradient. This procedure will separate thymidylate synthetase of cultures grown in D2O medium from thymidylate synthetase from cells grown in water medium. The presence of an intermediate enzyme band at 51% of the distance between the light and heavy bands has been taken as tentative evidence for the spontaneous formation of a light-heavy hybrid enzyme. After the shift from 37° to 25°, two major bands of thymidylate synthetase activity were found, one corresponding to light enzyme and one corresponding to heavy enzyme. From these data it was concluded that most of the thymidylate synthetase polypeptide chain had been synthesized at 37° and that the chain was completed with light amino acids at 25°. A model is presented proposing that thy-101 at nonpermissive temperatures is uniquely blocked in the synthesis of thymidylate synthetase because of an amino acid change which prevents the growing polypeptide chain from assuming the proper conformation and that this defect inhibits further movement of the ribosome.