Transcription of bacteriophage T4 late genes requires concomitant DNA replication. T4 late promoters, which consist of a single 8-bp –10 motif, are recognized by a holoenzyme containing Escherichia coli RNA polymerase core and the T4-encoded promoter specificity subunit, gp55. Initiation of transcription at these promoters by gp55-holoenzyme is inefficient, but is greatly activated by the DNA-loaded DNA polymerase sliding clamp, gp45, and the coactivator, gp33. We report that gp33 attaches to the flap domain of the Escherichia coli RNA polymerase β-subunit and that this interaction is essential for activation. The β-flap also mediates recognition of –35 promoter motifs by binding to σ 70 domain 4. The results suggest that gp33 is an analogue of σ 70 domain 4 and that gp55 and gp33 together constitute two parts of the T4 late σ. We propose a model for the role of the gp45 sliding clamp in activation of T4 late-gene transcription.