Reconstituted Transcription System Research Articles

CCAAT Binding Factor (CBF) Binding Mediates Cell Cycle Activation of Topoisomerase IIα: CONVENTIONAL CBF ACTIVATION DOMAINS ARE NOT REQUIRED

To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase II alpha (topo II alpha) promoter, which is activated during the late S and G(2)/M phases of the cell cycle and contains multiple CBF binding sites. Mutational analysis of the promoter shows that CBF binding to an inverted orientation of the CCAAT motif in the topo II alpha promoter, but not to a direct orientation, is required for transcription activation during the cell cycle. In contrast, analysis of the promoter in an in vitro reconstituted transcription system shows that CBF activates transcription of the topo II alpha promoter irrespective of the orientation of the CBF binding sites. This analysis demonstrates that only one of the three transcription start sites of the topo II alpha promoter is stimulated by CBF, indicating that transcription activation by CBF is dependent on basal promoter structure. Interestingly, mutations of the start site that abolish CBF-dependent transcription activation in vitro do not inhibit activation of the promoter during the cell cycle. Consistent with this observation, expression of a truncated CBF-B subunit lacking a transcription activation domain, which inhibits activity of a collagen promoter, does not affect activity of the topo II alpha promoter in fibroblast cells. In contrast, expression of an allele-specific CBF-B mutant that binds high affinity to a mutant CBF binding site containing a CCAAC motif revives transcription activation of an inactive mutant topo II alpha promoter containing CCAAC during the cell cycle. Altogether, this study indicates that CBF binding, but not conventional CBF activation domains, are required for activation of the topo II alpha promoter during the cell cycle. Considering these results together with results of another recent study, we hypothesize that binding of CBF that disrupts the nucleosomal structure in the topo II alpha promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo II alpha promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.

CBF/NF-Y functions both in nucleosomal disruption and transcription activation of the chromatin-assembled topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in chromatin is dependent on the promoter structure.

To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.

Human Taf(II)130 is a coactivator for NFATp.

NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAF(II)) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein (TBP) alone was insufficient in supporting activated transcription. In vitro interaction assays revealed that human TAF(II)130 (hTAF(II)130) and its Drosophila melanogaster homolog dTAF(II)110 bound specifically and reproducibly to immobilized NFATp. Sequences contained in the C-terminal domain of NFATp (amino acids 688 to 921) were necessary and sufficient for hTAF(II)130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAF(II)250, and hTAF(II)130 supported NFATp-activated transcription, demonstrating the ability of hTAF(II)130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAF(II)130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAF(II)130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.

TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

BackgroundNFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp.ResultsWe developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain.ConclusionsWe conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism.

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription.

Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system. Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p. Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells. Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I-Rrn3p complex and were inactive in promoter-dependent transcription. Activity was restored by adding purified pol I-Rrn3p complex to extracts from stationary phase cells. The pol I-Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation. We propose that the formation and disruption of the pol I-Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation.

Involvement of TFIID and USA components in transcriptional activation of the human immunodeficiency virus promoter by NF-kappaB and Sp1.

The purified Rel/NF-kappaB (p50/p65) complex and Sp1 markedly activate transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a highly purified HeLa reconstituted transcription system. Transcriptional activation by NF-kappaB and Sp1 requires both TFIID and the USA fraction. The USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator activity, both by repressing the basal level of transcription and by potentiating activator function to yield large increases in the levels of transcription induction. Under limiting concentrations, PC2 and PC4 also show synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65 subunit of NF-kappaB is a potent activator when assayed as a Gal fusion in the reconstituted transcription system and interacts both with TATA-binding protein (TBP) and with several human TBP-associated factors (TAFs) that include TAFII250. The p65 activation domain mediates transcription activation in the presence of partially reconstituted TFIID species that include a minimal complex containing only TBP and TAFII250. These studies also show that, like USA components, TAFs can serve both to repress TBP-mediated transcription and, following activator interactions, to reverse the repression and effect a net increase in activity. Taken together, these data underscore the importance of both TAFs and specific USA-derived coactivators for optimal activation of the HIV-1 promoter, as well as certain parallels in their overall mechanisms of action.

The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication.

The NS1 protein (139 amino acids) is one of the two nonstructural proteins of human respiratory syncytial virus (RSV) and is encoded by a very abundant mRNA transcribed from the promoter-proximal RSV gene. The function of NS1 was unknown and was investigated here by using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins (N, P, L and M2-1) expressed from separate cotransfected plasmids. Coexpression of the NS1 cDNA strongly inhibited transcription and RNA replication mediated by the RSV polymerase, even when the level of expressed NS1 protein was substantially below that observed in RSV-infected cells. The effect depended on synthesis of NS1 protein rather than NS1 RNA alone. Transcription and both steps of RNA replication, namely, synthesis of the antigenome and the genome, appeared to be equally sensitive to inhibition. The efficiency of encapsidation of the plasmid-derived minigenome was not altered by coexpression of NS1, indicating that the inhibition occurs at a later step. In two different dicistronic minigenomes, transcription of each gene was equally sensitive to inhibition by NS1. This suggested that the gradient of transcriptional polarity was unaffected and that the effect of NS1 instead probably involves an early event such as polymerase entry on the genome. NS1-mediated inhibition of transcription and RNA replication was not affected by coexpression of the M2 mRNA, which has two open reading frames encoding the transcriptional elongation factor M2-1 and the putative negative regulatory factor M2-2. The potent nature of the NS1-mediated inhibition suggests that negative regulation is an authentic function of the NS1 protein, albeit not necessarily the only one.

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

Increased Expression of the N Protein of Respiratory Syncytial Virus Stimulates Minigenome Replication but Does Not Alter the Balance between the Synthesis of mRNA and Antigenome

A popular model for RNA synthesis by nonsegmented negative-strand RNA viruses is that transcription and RNA replication are executed by the same polymerase complex and that there is a dynamic balance between the two processes that is mediated by the nucleocapsid N protein. According to this model, transcription occurs until sufficient soluble N protein accumulates to initiate encapsidation of the nascent RNA product, which somehow switches the polymerase into a readthrough replicative mode. This model was examined for respiratory syncytial virus (RSV) using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins that are expressed intracellularly from transfected plasmids. Preliminary experiments showed that reconstituted RNA replication was highly productive, such that on average each molecule of plasmid-supplied minigenome that became encapsidated was amplified 10- to 50-fold. N protein was increased on its own or in concert with the phosphoprotein P and in the presence or absence of the M2 ORF1 transcription elongation factor. The maximum level of N and P protein expression achieved from plasmids equalled or exceeded that obtained in RSV-infected cells. Increased levels of N protein stimulated RNA replication. This is consistent with the idea that RNA replication is dependent on the availability of N protein for encapsidation, which is one postulate of the model. The M2 ORF1 protein had no detectable effect on RNA replication under the various conditions of expression of N and P, which confirmed and extended previous results. However, there was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (synthesis of antigenome). The synthesis of positive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product.

Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein.

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.

Activation of transcription by progesterone receptor involves derepression of activation functions by a cofactor.

Hormone-induced progesterone receptors (PR) bound to response elements stimulate transcription initiation at target promoters through a mechanism that presumably involves cofactors or coactivators. To allow identification of such cofactors of transcriptional activation in a functional assay, we have established a reconstituted transcription system that is characterized by a specific loss of responsiveness to purified baculovirus-expressed wild type PR. In contrast to wild type PR, a C-terminally truncated PR mutant displayed strong activation potential in this system. As the purified recombinant full-length PR is capable of DNA binding, our results suggest that C-terminal sequences of PR mediate a cis-repression of N-terminal activation functions. Moreover, using this PR-nonresponsive transcription system, we identified and partially purified an activity from rat liver, termed COPRA (cofactor of PR activation), that restores transactivation by full-length PR. Characterization of COPRA revealed that this cofactor exhibits activator specificity and is not involved in basal transcription. We postulate that COPRA acts by relieving the repression of activation functions mediated by C-terminal sequences.

Analysis of the 3′ Terminal Sequence Recognized by the Rift Valley Fever Virus Transcription Complex in Its Ambisense S Segment

A reconstituted transcription system composed of the Rift Valley fever phlebovirus (Bunyaviridae family) proteins L and N expressed via recombinant vaccinia viruses and an S-like model RNA containing the CAT gene in the antisense orientation, has been described previously by Lopezet al.(J. Virol.,1995, 69, 3972–3979). We extended the use of thisin vivosystem to determine the sequence at the 3′ end of the ambisense S segment recognized by the transcription complex. A mutational analysis of the sequences at the 3′ end of the S-like genomic or antigenomic RNA was undertaken. The data indicated that the minimal sequence required for transcription resides in the 13 first 3′ nucleotides of the genomic or antigenomic RNA. In these sequences, two regions appeared crucial: the bases at positions 3 to 8 and the purine at position 13. In addition, the terminal repeat ...GU could be deleted without affecting significantly the template activity of the RNA. These data support the prime and realign mechanism proposed recently for Bunya- and Arenaviruses

Repression of RNA Polymerase II and III Transcription during M Phase of the Cell Cycle

Nuclear transcription is repressed when eukaryotic cells enter mitosis. UsingXenopusegg extracts shifted to the mitotic state with recombinant cyclin B1 protein, we have been able to reproduce mitotic repression of transcriptionin vitro.Active RNA polymerase III transcription is observed in interphase extracts in the absence of added cyclin, but is strongly repressed by the induction of cdc2/cyclin B (maturation/mitosis promoting factor, MPF) kinase activity in the mitotic extract. Studies with protein kinase inhibitors show that protein phosphorylation is required for repression. Add-back experiments indicate that repression of class III gene transcription is due to inactivation of the transcription factor TFIIIB. TFIIIB is composed of the TATA-box binding protein (TBP) and TBP-associated factors of 75 and 92 kDa. In the present study, we show that TBP and a polypeptide of 92 kDa are substrates of the mitotic kinase in highly purified TF- IIIB fractions. We also show that a phosphatase present in theXenopusegg extract can reactivate transcription after repression by the mitotic kinases. This result suggests a mechanism for reactivation of transcription after exit from mitosis into the G1 phase of the cell cycle. As for pol III genes, purified cdc2/cyclin B kinase is sufficient to inhibit transcription by RNA polymerase II in a reconstituted transcription system containing the basal transcription factors and polymerase.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

TFIIS binds to mouse RNA polymerase I and stimulates transcript elongation and hydrolytic cleavage of nascent rRNA.

Efficient transcription elongation by RNA polymerase I (Pol I) requires a specific Pol I-associated factor, termed TIF-IC. Here we show that TFIIS, a factor that has previously been shown to promote read-through past many types of blocks to elongation by RNA polymerase II, also enhances Pol I-directed transcription elongation. In a reconstituted transcription system containing purified proteins, TFIIS stimulates Pol I transcription by increasing the overall rate of RNA chain elongation. As with Pol II, ternary Pol I complexes cleave the 3' end of the nascent transcripts in response to TFIIS. The truncated RNAs remain bound to the template, are subject to pyrophosphorolysis, and can be chased into longer transcripts. Moreover, we show by immunoprecipitation and specific affinity chromatography that TFIIS physically interacts with Pol I. The results suggest that nascent transcript cleavage by TFIIS or a TFIIS-related factor may be a general mechanism by which both Pol I and Pol II can bypass transcriptional impediments.

Transcriptional Corepression In Vitro: a Mot1p-Associated Form of TATA-Binding Protein Is Required for Repression by Leu3p

Signals from transcriptional activators to the general mRNA transcription apparatus are communicated by factors associated with RNA polymerase II or the TATA-binding protein (TBP). Currently, little is known about how gene-specific transcription repressors communicate with RNA polymerase II. We have analyzed the requirements for repression by the saccharomyces cerevisiae Leu3 protein (Leu3p) in a reconstituted transcription system. We have identified a complex form of TBP which is required for communication of the repressing signal. This TFIID-like complex contains a known TBP-associated protein, Mot1p, which has been implicated in the repression of a subset of yeast genes by genetic analysis. Leu3p-dependent repression can be reconstituted with purified Mot1p and recombinant TBP. In addition, a mutation in the Mot1 gene leads to partial derepression of the Leu3p-dependent LEU2 promoter. These in vivo and in vitro observations define a role for Mot1p as a transcriptional corepressor.

Species Specificity of Ribosomal Gene Transcription: A Factor Associated with Human RNA Polymerase I Prevents Transcription of Mouse rDNA

An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF-IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species-specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB-directed initiation complexes at the murine rDNA promoter.

Characterization of the Components of Reconstituted Saccharomyces cerevisiae RNA Polymerase I Transcription Complexes

We have reconstituted specific RNA polymerase I transcription from three partially purified chromatographic fractions (termed A, B, and C). Here, we present the chromatographic scheme and the initial biochemical characterization of these fractions. The A fraction contained the RNA polymerase I transcription factor(s), which was necessary and sufficient to form stable preinitiation complexes at the promoter. Of the three fractions, only fraction A contained a significant amount of the TATA binding factor. The B fraction contributed RNA polymerase I, and it contained an essential RNA polymerase I transcription factor that was specifically inactivated in response to a significant decrease in growth rate. The function of the C fraction remains unclear. This reconstituted transcription system provides a starting point for the biochemical dissection of the yeast RNA polymerase I transcription complex, thus allowing in vitro experiments designed to elucidate the molecular mechanisms controlling rRNA synthesis.