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...

The DNA sequences required for faithful initiation of ribosomal RNA transcription were determined. BAL-31 digestion was used to modify the rDNA template by introducing deletions from its 3'- and 5'-ends. The resulting mutant DNAs were tested for template activity individually or in competition with wild type utilizing an in vitro transcription system from Acanthamoeba castellanii. The results identify the sequence extending from -31 to +8 to be absolutely required for transcription. In addition; when the region between -47 and -32 is left intact, transcription is augmented.

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The binding of a species-specific transcription initiation factor (TIF) and purified RNA polymerase I to the promoter region of the 39S ribosomal RNA gene from Acanthamoeba were studied by using DNase I "footprinting." Conditions were chosen such that the footprints obtained could be correlated with the transcriptional activity of the TIFcontaining fractions used and that the labeled DNA present would itself serve as a template for transcription. The transcription factor binds upstream from the transcription start site, protecting a region extending from around -14 to -67 on the coding strand, and -12 to -69 on the noncoding strand. The protein that binds to DNA within this region can be competed out by using wild-type promoters but not by using mutants which do not stably bind the factor. RNA polymerase I can form a stable complex in the presence of DNA and transcription factor, allowing footprinting of the complete transcription initiation complex. RNA polymerase I extends the protected region obtained with TIF alone to around + 18 on the coding strand, and to +20 on the noncoding strand. This region is not protected by polymerase I in the absence ofTIF. The close apposition of the regions protected by TIF and polymerase provides evidence that accurate transcription of the ribosomal gene may be achieved through protein-protein contacts as well as through DNA-protein interactions.Transcription initiation of eukaryotic genes in vitro requires the presence of at least one protein factor in addition to RNA polymerase and a promoter-containing DNA fragment (1-3). For class II, III, and possibly class I genes, one or more of the transcription factors acts through stable interaction with the gene promoter regions (4-6), allowing correct initiation by the polymerase. The details of this process differ considerably between different gene classes, with respect to the sequence and positioning of promoter regions as well as the number of factors thought to be required for transcription.Control regions for polymerase II, and to some extent polymerase III, show promoter sequence homology when comparisons between species are made (7,8). In contrast, the promoter sequences involved in polymerase I transcription are highly diverged, making identification of regulatory sequences by comparison of conserved regions difficult.Studies on ribosomal gene promoters do, however, show that a region flanking the 5' side of the initiation start site is required for transcription (9). Part of this region functions by interaction with protein components of the system to form a stable complex that commits the template for correct transcription (6,(10)(11)(12). In the Acanthamoeba rRNA genes, the sequence required for template commitment extends from around -20 to -47, and it can be divided into two regions: one (A region) is absolutely required for transcription, and the other (B region) is involved with the stability of a preinitiation complex formed between the DNA and a transcription initiation factor (TIF) (6). A third region fla...

Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decreased specific RNA synthesis in vitro. These were within the region which is protected from DNase I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.Initiation of specific gene transcription by RNA polymerase (RNAP) in both procaryotic and eucaryotic cells requires interaction between a protein and specific nucleotide sequences on the DNA template, the promoter (5,6,20,21,36). In procaryotes, a specific subunit of the single omniscient polymerase functions in this role. In contrast, there are three distinct polymerases in eucaryotes: RNAP I, II, and III, responsible for pre-rRNA, heterogeneous nuclear RNA, and pre-tRNA plus 5S RNA biosynthesis, respectively (32). Each requires the interaction of specific trans-acting protein factors with the promoter in addition to RNAP for initiation. More so than RNAP II and RNAP III, faithful initiation of rRNA transcription shows species specificity, i.e., there is very poor cross-reactivity between transcription extracts and promoters among different species (12). This specificity has been shown to reside in the trans-acting protein which interacts with the promoter DNA (25). The nucleotide sequences of the RNAP I promoters from humans (19,24), mice (7,11,37,38), rats (28), Xenopus spp. (22,35), Drosophila melanogaster (16), and lower eucaryotic organisms (4, 18, 29, 34) have been reported. In apparent agreement with the notion that species specificity is elicited by sequence-specific binding of trans-acting proteins, these promoters showed no, or only low, homology except for those closely related species such as mouse and rat (10), and these show relaxed species selectivity (reviewed in reference 36).Because of the sequence divergence of rRNA promoters between organisms and the species specificity of the transcription initiation factor (TIF), a number of rRNA promoters are being examined to discern motifs with common functions rather than homologous primary sequences (36).We used an in vitro rRNA transcription system from * Corresponding author.