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.

We have utilized a cell-free transcription system from Acanthamoeba castellanii to test the functional activity of RNA polymerase I and transcription initiation factor I (TIF-I) during developmental down regulation of rRNA transcription. The results strongly suggest that rRNA transcription is regulated by modification, probably covalent, of RNA polymerase I: (1) The level of activity of TIF-I in extracts from transcriptionally active and inactive cells is constant. (2) The number of RNA polymerase I molecules in transcriptionally active and inactive cells is also constant. (3) In contrast, though the specific activity of polymerase I on damaged templates remains constant, both crude and purified polymerase I from inactive cells have lost the ability to participate in faithful initiation of rRNA transcription. (4) Polymerase I purified from transcriptionally active cells has the same subunit architecture as enzyme from inactive cells. However, the latter is heat denatured 5 times faster than the active polymerase.

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...

Proper initiation of transcription by RNA polymerase II requires the TATA-consensus-binding transcription factor TFIID. A cDNA clone encoding the Drosophila TFIID protein has been isolated and characterized. The deduced amino acid sequence reveals an open reading frame of 353 residues. The carboxyl-terminal 180 amino acids are approximately 80% identical to yeast TFID and 88% identical to human TFIIID. The amino-terminal portions of the yeast and Drosophila TFIID proteins lack appreciable homology, whereas the Drosophila and human amino termini appear qualitatively similar. In addition, the amino-terminal region of the Drosophila TFID contains several sequence motifs that are found in other Drosophia proteins which appear to regulate transcription. (6)(7)(8). One key general transcription factor is TFIID, which binds to the TATA element in Drosophila (9), human (10, 11), and yeast (12)(13)(14). TFIID has been shown to be required for preinitiation complex formation, suggesting that it functions at an early critical step in the initiation process (15, 16). The yeast and human TFIID proteins function interchangeably to support a basal level oftranscription in vitro, implying significant structural conservation between these molecules (12)(13)(14).The recent cloning ofthe yeast (Saccharomyces cerevisiae) TFIID gene has allowed an examination of the functional and putative structural properties of this transcription factor (17-21 A Drosophila melanogaster embryonic (0-24 hr) cDNA library in AZAP II (Stratagene) was screened by hybridization with a cloned PCR probe. Hybridizations were performed at 400C in 6x SSPE/0.25% nonfat dried milk/50%o deionized formamide. Filters were washed at 650C in 2x SSC (lx = 0.15 M NaCl/15 mM sodium citrate, pH 7.0)/0.1% NaDodSO4. After plaque purification, individual cDNA inserts were recovered in the form of chimeric pBluescript SK(+) phagemids by in vivo excision from the A vector as described by the library supplier.Overproduction of Recombinant Drosophila TFVD. The conserved region ofDrosophila TFIID was subcloned into an Escherichia coli expression system as follows. The Apa I-EcoRI fragment of the Drosophila TFIID cDNA insert encoding the carboxyl-terminal 1% amino acid residues was inserted into the pET-8c (T7 expression) vector (27) at the Nco I-BamHI restriction sites. A BamHI site was added to the EcoRI site of the TFIID insert by ligation of an EcoRIBamHI (duplex oligonucleotide) adaptor (New England Biolabs). The modified TFIID insert was then ligated into pET-8c cut with BamHI and Nco I. The 3' overhang of the TFIID Apa I site was joined to the 5' overhang of the Nco I site of pET-8c by use of an eight-base "bridging oligonucleotide" with sequence complementarity to both the Nco I and Apa I overhangs (5'-CATGGGCC-3'). The trimolecular ligation joined the initiating methionine codon of the pET-8c vector in-frame to the Gly-158 codon of the TFIID open reading frame. Plasmid DNA samples prepared from XL1-Abbreviations: PCR, polymerase chain reaction; IPTG, isopropy...