USE OF A MACROMOLECULAR CROWDING AGENT TO DISSECT INTERACTIONS AND DEFINE FUNCTIONS IN TRANSCRIPTIONAL ACTIVATION BY A DNA-TRACKING PROTEIN- BACTERIOPHAGE-T4 GENE-45 PROTEIN AND LATE TRANSCRIPTION

We have used a molecular crowding reagent to define functions in the t ranscriptional activation of bacteriophage T4 late genes. This activat ion normally requires the three T4 DNA polymerase accessory proteins e ncoded by T4 genes 44, 62, and 45 (the gp44/62 complex and gp45), an e nhancer-like cis-acting site, an RNA polymerase-bound coactivator, and an unobstructed path along the DNA joining the promoter to the enhanc er. We show that molecular crowding eliminates the requirement for the gp44/62 complex and for the enhancer, retains the requirement for gp4 5 and its coactivator, and generates activated promoter complexes with nearly unchanged DNase I footprints. These experiments identify gp45 as the direct activator of transcription, and the gp44/62 complex as t he assembly factor for gp45. They suggest that the enhancer serves as the normal, but not invariably essential, entry site for the gp45 DNA- tracking protein.