Conditions allowing synthesis of thymidylate synthetase at the nonpermissive temperature in a temperature-sensitive thy mutant normally blocked in its translation.

Escherichia coli thyAl is a temperature-sensitive mutant altered in the structural gene for thymidylate synthetase. A previous study from this laboratory has shown that thyAl 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 thyAl cultures can carry out net synthesis of thymidylate synthetase at the nonpermissive temperature. A thyAl 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 Thyphenotype 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 D20 medium at 25” and Hz0 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 thyAl 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.