The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesizes the large rRNA, pol II synthesizes mRNA and pol III synthesizes tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences.

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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...

An in vitro transcription system consisting of partially purified transcription initiation factor(s) and purified RNA polymerase I from Acanthamoeba casteUanii was used to study the mechanism of faithful initiation of ribosomal RNA transcription. Formation of a preinitiation complex between one or several auxiliary transcription proteins and the DNA template in the absence of RNA polymerase I was demonstrated. A series of 3'-and 5'-deletion mutants of the template was used in prebinding competition experiments and provided evidence for three distinct functional regions of the promoter: core motif A interacts with the transcription initiation factor(s) and is required for faithful transcription; the start motif is required for transcription, but it can be deleted without affecting the binding of transcription initiation factor(s); and motif B stabilizes preinitiation complex formation (in addition to core motif A), but it is dispensable for faithful initiation of transcription. (8) demonstrated that it is the species-specific TIF that is involved in preinitiation complex formation. Evidence was also reported supporting the notion that RNAP-I was not needed for preinitiation complex formation (8). However, since the polymerase preparation used in their study also formed a stable complex with the DNA template, the role of RNAP-I in complex formation was unclear. In contrast, we have used a partially purified TIF preparation and highly purified RNAP-I from the protozoan Acanthamoeba castellanii, incapable of specific initiation in vitro (11). Using these preparations, we demonstrate that, in analogy to polymerase II and III systems, the TIFs first bind to the promoter in the absence of RNAP-I to form a stable preinitiation complex. RNAP-I then binds to this complex to form an initiation complex capable of de novo synthesis of a faithful RNA transcript. In addition, we have used a series of deletion mutants to identify the template sequences involved in complex formation.The core promoter (which we define as the minimal DNA sequence required for faithful in vitro transcription) was shown to consist of two sequence motifs. One motif is proximal to the start site and is required for transcription but not for TIF binding. Two upstream sequences are involved in TIF binding. Only one is required for transcription and transiently interacts with TIF; the second is necessary for stable preinitiation complex formation. MATERIALS AND METHODSDNA Templates. A 74-base-pair (bp) Xma III-generated fragment containing the initiation region for the ribosomal RNA gene of Acanthamoeba was cloned into the Xma III site of pBR322. This ribosomal DNA fragment, extending from -55 to +19, was inserted in both orientations to produce the clones pSBX60 and pSBX60i. In the following experiments, the plasmids were linearized with different restriction enzymes (Bethesda Research Laboratories) to produce RNA runoffs of diverse size in the cell-free transcription system (Fig. 1). pSBX60 (3' deletions) or pSBX60i (5' deletions) were cut wit...