Conditions Allowing Synthesis of Thymidylate Synthetase at the Nonpermissive Temperature in a Temperature-sensitive thy Mutant Normally Blocked in Its Translation

Abstract

Abstract Escherichia coli thyA101 is a temperature-sensitive mutant altered in the structural gene for thymidylate synthetase. A previous study from this laboratory has shown that thyA101 belongs to a new class of temperature-sensitive mutants (Group IV) and is blocked in the synthesis of thymidylate synthetase at the level of the polyribosome complex. The block has been postulated to result from a conformational defect in the growing polypeptide chain of thymidylate synthetase. On shifting to the permissive temperature, the chain assumes the proper configuration and the enzyme is completed by a process requiring protein synthesis. This study describes conditions under which thyA101 cultures can carry out net synthesis of thymidylate synthetase at the nonpermissive temperature. A thyA101 culture grown at 25° and then shifted to 37°, the nonpermissive temperature, continues to form thymidylate synthetase, although more slowly than other proteins. Ultimately the enzyme is diluted during cell division and the Thy- phenotype is expressed in the form of thymineless death. Synthesis of thymidylate synthetase at 37° after shifting from 25° requires protein synthesis. Furthermore protein synthesis must also occur in the prior incubation at 25°. Density and temperature shift studies in D2O medium at 25° and H2O medium at 37° and the use of CsCl gradients to analyze for heavy and light enzyme activity have demonstrated that new thymidylate synthetase is synthesized at 37°. From these studies we have suggested a model for thyA101 in which thymidylate synthetase (or its subunits) previously formed at 25° interacts at 37° at the level of the polyribosome complex with the defective and blocked polypeptide chain to effect a conformational change which allows the protein to be completed. It was suggested that in this type of rescue the preformed subunit be considered as a conformational template.