It has been generally accepted that the TATA binding protein (TBP) is a uni
versal mediator of transcription by RNA polymerase I, II, and III. Here we
report that the TBP-related factor TRF1 rather than TBP is responsible for
RNA polymerase III transcription in Drosophila. Immunoprecipitation and in
vitro transcription assays using immunodepleted extracts supplemented with
recombinant proteins reveals that a TRF1:BRF complex is required to reconst
itute transcription of tRNA, 5S and U6 RNA genes. In vivo, the majority of
TRF1 is complexed with BRF and these two proteins colocalize at many polyte
ne chromosome sites containing RNA pol III genes. These data suggest that i
n Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a ma
jor role in RNA pol III transcription.