The role of the Acanthamoeba casteUlanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.While the transcription systems of eukaryotic RNA polymerases I, II, and III obviously share some characteristics, initiation mechanisms for these transcription systems have been largely studied separately. The polymerases themselves have five subunits in common, seven in the case of the "odd pols," RNA polymerases I and III (45). Nevertheless, the general transcription factors involved with each polymerase have been examined in isolation, perhaps masking important generalizations about their functions. This approach began to change when it was discovered that some of the genes for small nuclear RNAs (snRNAs) are transcribed by polymerase III whereas most are transcribed by polymerase II (27,32,35,47 The full impact of this factor overlap was perhaps not realized because polymerase I still appeared to use dedicated factors. However, a flurry of studies (6,7,44,56) revealed that TBP was required for transcription of all genes. TBP is now known to be a subunit of TFIID (41), TFIIIB (17,24,48,55), and human TIF-IB (SL1) (6). TBP is associated with additional subunits (TAFs) to make up the functional factors (reviewed in reference 41). The TAFs appear to be different for each polymerase system (reviewed in reference 42), although overlap of TAFs has not been rigorously ruled out. All the TBP-containing factors are pivotal for their respective polymerases. Indeed, TFIIIB and TIF-IB are fundamental transcription factors; i.e., they appear to be responsible for the repetitive recruitment of RNA polymerase during successive rounds of initiation (16; reviewed in references 36 and 37).The manner by which the TBP-containing factor is recruited to the promoter differs from gene to gene. For RNA polymerase III genes with type I (SS RNA) or type II (tRNA, VAI, Alu, EBER, 7SL, 4.5S) internal control regions, additional general transcription factors are required to assemble TFIIIB onto the promoter (reviewed in references 10-12). Initiation complex formation on 5S and tRNA genes is an organized process in which the factors bind in an obligatory order, each relying on protein-DNA and protein-protein interactions with a previously bound factor(s) to join the complex. On some genes the DNA interaction site for TFIIIB is sequence specific, whereas on others specific sequence recognition is no...