TUF, the yeast DNA-binding factor specific for UASrpg upstream activating sequences: identification of the protein and its DNA-binding domain.

Abstract

The factor TUF interacts specifically with RPG or HOMOL1 sequences, which are present upstream of many genes coding for the yeast translational apparatus. Here we present evidence that the RPG and HOMOL1 motifs are variants of a consensus UASrpg (upstream activating sequence) recognized by the same factor. Factor TUF was identified by using two highly selective methods. (i) The DNA-protein complex was isolated by pore-limit electrophoresis in polyacrylamide gradient gels and found to contain a single polypeptide fo 150 kDa. (ii) In a two-step protein-blotting/nuclease-protection ("footprinting") procedure, the same 150-kDa polypeptide blotted on nitrocellulose exhibited the same specific DNA-binding properties as TUF factor. A 50-kDa DNA-binding domain of TUF was isolated by selective proteolysis. This suggests a bipolarization of the TUF protein, with distinct functional domains.