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Volume 270, Number 11, Issue of March 17, 1995 pp. 6205-6210
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Components of Reconstituted Saccharomyces cerevisiae RNA Polymerase I Transcription Complexes (*)

(Received for publication, November 8, 1994; and in revised form, January 11, 1995)

Daniel L. Riggs (§),  ,  Cheryl L. Peterson ,  J. Quyen Wickham ,  Letrisa M. Miller ,  Eileen M. Clarke ,  John A. Crowell ,  Jean-Christophe Sergere

From the Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.

INTRODUCTION

In rapidly growing cells, rRNA synthesis is one of the largest single consumers of cellular resources. In these cells, the high level of rRNA synthesis is required to produce the number of ribosomes necessary to meet the translational load. In contrast, cells growing at a slower rate do not require the same level of protein synthesis. One would predict that these cells would conserve energy by reducing the rate of rRNA synthesis and ribosome biogenesis, resulting in fewer ribosomes per cell. Indeed, this has been demonstrated in both procaryotic and eucaryotic organisms, although the mechanisms involved remain quite obscure in all cases.

In eucaryotes, the bulk of rRNA synthesis is catalyzed by RNA polymerase I (RNAP I). (^1)Three RNAP I ancillary factors have been described in a number of different systems. These factors, and their nomenclature in various systems, have been recently reviewed(1) . The best characterized factor is a rather nonspecific DNA binding protein termed Ubf (upstream binding factor). The identification and isolation of the genes encoding Ubf from these organisms has facilitated detailed structural and functional studies of this protein. Ubf binds rather promiscuously to DNA, although it does produce a distinct footprint on both the promoter (primarily the upstream element) and enhancer regions from several organisms(2, 3, 4) . It has been shown that Ubf may not be absolutely required for transcription in vitro under specific conditions but rather it acts as a stimulatory factor(4, 5, 6) . The possible role of Ubf as a facilitator of transcription complex formation is also supported by recent experiments that show that Ubf induces considerable conformational changes in the promoter structure (7, 8, 9) .

The second widely recognized RNAP I transcription factor, termed SL-1 in human systems, contains several polypeptides, and it appears to form the core of the transcription complex. Independently, this protein complex has a variable DNA binding activity. Human SL-1 does not exhibit any specific DNA binding properties(2) , although the rat and mouse homologues can bind the promoter(4, 10) . A major breakthrough in the characterization of SL-1 was the discovery that it contained the TATA binding protein (TBP), along with a number of other associated proteins termed TATA-associated factors(11) . TBP was originally identified as a component of the RNAP II transcription factor IID, and it was subsequently found to be involved in all three nuclear transcription systems(12, 13) .

rRNA synthesis in vivo is responsive to a wide variety of agents or treatments that alter cellular growth or protein synthesis. In a number of cases, extracts isolated from these treated cells exhibit the regulatory response observed in vivo; that is, they have altered levels of specific RNAP I transcription (reviewed in (1) and (14) ). In these cases, this response is due to the inactivation of a factor found associated with RNAP I. This factor, known as C*, TIFI-A, or TFIC(15, 16, 17, 18) , is necessary for formation of the initiation complex and is inactivated early in the transcription cycle(16, 19, 20) . Several lines of evidence suggest that regulation of the activity of the RNAP I-associated factor may not be the only mechanism by which rRNA synthesis is regulated. Additional targets of regulation may include Ubf (21, 22, 23) or an RNAP I-specific inhibitor (24) .

While the reconstitution of enzymatic activity in vitro has been a valuable tool for a functional dissection of these macromolecular structures, the yeast RNA polymerase I system has been much less amenable to this type of analysis. Several protocols for the preparation of small scale yeast RNAP I transcription extracts have been described(25, 26, 27) . These extracts have been useful for delineating the structure and function of cis-acting elements, but the inherent limitations of their scale have precluded a detailed biochemical analysis of trans-acting factors. Yeast is a potentially very powerful system in which to study rRNA synthesis because of well defined genetics, as well as the relative ease with which one can rapidly alter cell growth rate through precise, chemically defined nutritional changes. The potential of this system has yet to be fully realized in large part because of difficulties in biochemically defining the RNAP I transcription apparatus. Here, we report the development of a protocol to prepare large quantities of very active transcription lysates and their use in an initial biochemical identification and characterization of the yeast RNAP I trans-acting factors. This work provides a starting point for the purification and complete characterization of the components of the yeast RNAP I transcription complex, which, in conjunction with genetic analysis, will help us to understand the molecular mechanisms that regulate rRNA synthesis.

MATERIALS AND METHODS

Plasmids

The plasmid pDR10 contains the RNAP I promoter on an rDNA PvuII fragment (from -1478 to +582 relative to the start point of RNAP I transcription) inserted into the SmaI site of pBluescript II KS+ (Stratagene). This construct also contains the 5 S rRNA gene, which is transcribed by RNAP III. In some experiments, the 5 S rRNA gene template pBB111R (28) was used.

Growth of Cultures

The yeast strain JHRY20-2CDelta1 (29) was cultured at 30 °C in YEPD (1% w/v yeast extract, 2% w/v peptone, and 2% w/v glucose) with the pH adjusted to 5.5 with HCl. The medium was inoculated with a low density overnight culture (optical density at 595 nm, A, of less than 3.0) resulting in an initial density of 0.1-0.2 A units. The culture was incubated at 30 °C for at least three generations in exponential phase (generation time of 1.5 h): then, the cells were harvested when the culture density reached an A of 1-2 (corresponding to approximately 5 times 10^7 cells/ml). For the preparation of transition and stationary phase cultures, the growth medium was supplemented with ampicillin (50 µg/liters) to discourage bacterial growth, and the cultures were vigorously aerated. The transition phase cells were harvested at a point when the culture was still growing but at a reduced growth rate of about 20 h per generation. Stationary phase cells were harvested 30 h after growth had ceased.

Cell Breakage

All manipulations were performed at 0-4 °C with ice-cold solutions. The buffers all contained 10 mM beta-mercaptoethanol and 0.1 mM phenylmethylsulfonyl fluoride unless otherwise noted. The cells were resuspended in solubilization buffer (0.2 M Tris acetate, pH 7.5, 10% (v/v) glycerol, 10 mM magnesium acetate, 0.4 M ammonium sulfate) at a final concentration of 0.5 g of cells/ml. For the large scale lysates, approximately 80-100 g (wet pellet weight) of cells were lysed using a Bead Beater (Bio-Spec, Bartlesville, OK) in a 300-ml chamber filled with 180 ml of beads (0.5 mm in diameter). Sufficient additional solubilization buffer was added to the chamber to exclude all of the remaining air. The cells were beaten for 30 s followed by several minutes of rest in an ice/ethanol bath. Typically, 12-16 cycles were needed for adequate breakage (50-75% breakage as determined by phase contrast microscopy). The cell lysate was removed, and the beads were washed with solubilization buffer. The combined lysate and washes (about 280 ml, or 3 ml/g of cells) were centrifuged at 210,000 times g for 2 h. After the high speed spin, all of the supernatant was decanted and used for the subsequent steps. No attempt was made to pull off only the clear intermediate layer(26) .

The only modifications made for the preparation of small scale lysates concerned the breakage. The cells were broken in 8-ml plastic screw top tubes (for example, Sarstedt, Newton NC; 60.542PP) containing 3 ml of glass beads and several large beads (5 mm in diameter). The resuspended cells were added to the vial, the trapped air bubbles removed, and the vial sealed with the cap. The cells were lysed by vortexing as previously described(26) .

Precipitation of Extract

The high speed supernatant from the ultracentrifugation (either small or large scale), which was in 10% saturated 400 mM ammonium sulfate, was adjusted to 60% saturation by adding solid ammonium sulfate and incubated on ice for 30 min. After centrifugation (15,000 times g for 15 min), the 10-60% pellet was resuspended in a minimal volume of TA buffer (20 mM Tris acetate, pH 7.5, 10% (v/v) glycerol, 10 mM magnesium acetate) containing no KCl (here referred to as TA-0). The protein concentration was about 20-30 mg/ml, as determined using the Bradford dye binding procedure (30) . The residual ammonium sulfate was removed from this sample by extensive dialysis against TA-0 buffer. This sample was then diluted to about 10 mg/ml protein with TA-0 buffer and centrifuged at 10,000 times g for 10 min. Typically, the resulting pellet contained 10-25% of the total protein present in the 60% ammonium sulfate cut. When making small scale lysates, both the ``low salt pellet'' and ``low salt supernatant'' were retained for further analysis.

The low salt pellet, when resuspended, contained a significant amount of specific RNAP I activity as determined by in vitro transcription assays. To assay activity, this pellet was resuspended in a small volume of TA buffer containing 200 mM potassium glutamate (TA-200 KGlu). This suspension had a protein concentration of about 5 mg/ml, and typically 15 µg of this suspension was used in each transcription assay. The low salt supernatant contained a significant amount of RNAP III-specific transcriptional activity. To assay this material, this supernatant was first dialyzed against the transcription buffer TA-200 KGlu. Typically, the supernatant had a protein concentration of 10 mg/ml, and 50 µg of protein was added to the RNAP III transcription assay. The low salt pellets and supernatants were used to determine the levels of specific RNAP I and III transcriptional activity present in the cells that had been grown through transition phase into stationary phase.

For large scale RNAP I lysates, the low salt pellet was first solubilized in TA buffer containing 400 mM ammonium sulfate and then dialyzed against TA-0 to a conductivity equivalent to TA buffer containing 100 mM KCl (TA-100 KCl). After adjustment of the conductivity, the insoluble material, usually about half of the total protein, was removed by centrifugation (10,000 times g for 10 min). It was necessary to solubilize the low salt pellet in 400 mM ammonium sulfate instead of 100 mM KCl as the RNAP I components in the low salt pellet remained partially insoluble in TA-100 KCl buffer.

Q Gradient Chromatography

The supernatant was applied to a 10-ml column containing Macro-Prep high Q anion exchange support (Bio-Rad) at the rate of 3 mg of protein/ml of column bed volume. The loaded column was first washed with TA-100 KCl, and then the column was developed with a gradient from 100 to 700 mM KCl over 5 column volumes. Approximately 100 0.5-ml fractions were collected. The A, B, and C activities each eluted in 3-5 fractions, with 10-15 fractions separating adjacent activities.

Transcription Assays

Specific RNAP I transcription assays contained 20 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 200 mM potassium glutamate, 1.25 µg/ml creatine kinase, 30 mM creatine phosphate, 450 µM ATP, 200 µM UTP, 200 µM CTP, 15 µM [alpha-P]GTP (20-40,000 cpm/pmol, 5-10 µCi/40 µl assay), and 2.5-4 µg/ml DNA template (pDR10 linearized with EcoRV unless indicated) in a final volume of 40 µl. The extracts were preincubated with the DNA template for 5 min at 30 °C in the complete transcription assay mix without the G, U, and C nucleotides. Transcription was initiated by the addition of the omitted nucleotides. The reaction was then incubated for 5 min at 30 °C, after which it was stopped by extraction with phenol/chloroform. The RNA was precipitated with 2.5 volumes of ethanol containing 1 M ammonium acetate. The dried pellets were then resuspended in 3 µl of a denaturing loading buffer and electrophoresed on a 5% denaturing polyacrylamide gel (37.5:1 acrylamide:bisacrylamide). The dried gels were exposed to x-ray film with an intensifying screen at -70 °C, typically for several hours.

Nonspecific RNAP assays were performed under the same conditions, except that sheared salmon sperm DNA (100 µg/ml) replaced the specific transcription template and the final volume of each assay was 20 µl. The assays were terminated by the addition of phenol/chloroform and 10 µg of salmon sperm carrier DNA. Radiolabeled transcripts were precipitated with 2.5 volumes of ethanol containing 1 M ammonium acetate, and then the pellet was resuspended in 20 µl of water. A total of three sequential precipitation-resuspension cycles were performed to remove unincorporated radioactive nucleotides. The final resuspended pellet was spotted onto a disc of DE81 filter paper (Whatman), dried, and counted in a scintillation counter. To determine the background cpm for each sample assayed, an identical reaction was performed in parallel, but the sample was not incubated; rather, it was immediately terminated with phenol/chloroform after the addition of nucleotides to the assay. The results of these background assays were consistently about 1% of the most active samples (300 cpm). Tagetitoxin (Tagetin) was obtained from Epicentre Technologies (Madison, WI), and 20 units were added per assay. The alpha-amanitin concentration in the assays was 75 µg/ml.

Western Analysis

The samples, containing approximately 20 µg of protein each, were separated by SDS-polyacrylamide gel electrophoresis with a discontinuous buffer system and then transferred to a nitrocellulose membrane using a semi-dry transfer system (Panther, Owl Scientific, Inc. Cambridge, MA). The transfer buffer consisted of 25 mM Tris-Cl, 192 mM glycine, pH 8.3, and 20% (v/v) methanol. Western blotting was done in TBS-T (20 mM Tris-Cl, pH 7.6, 500 mM NaCl, and 0.5% (v/v) polyoxyethylenesorbitan monolaurate), following the ECL protocol RPN 2106 (Amersham Corp.). The primary antiserum was polyclonal anti-yeast TFIID (UBI, Lake Placid, NY). The TBP standard was a cell extract prepared from an Escherichia coli strain, which overexpressed yeast TBP (kindly provided by Dr. Martin Schmidt). The identity of TBP in this extract was confirmed by Western analysis alongside purified TBP.

RESULTS

Preparation and Fractionation of the Cell Extracts

We have extensively modified a protocol developed for making small scale extracts (26) to enable us to prepare large quantities of very active extracts that are amenable to biochemical analysis (Fig. 1). Here, we describe the protocol and the initial biochemical characterization of a reconstituted yeast RNAP I transcription system.

Figure 1: The fractionation scheme with the nomenclature of the intermediate fractions. AS, ammonium sulfate.


Whole cell extracts were prepared from exponential phase cells, which had been harvested at least one generation before the end of exponential phase. Cells were broken using glass beads in a blender with typically about 50-75% of the cells being lysed as judged by phase contrast microscopy. The high molecular weight cell material was removed by ultracentrifugation (210,000 times g for 2 h) as has been done for the yeast RNAP II transcription system(33) . The cleared supernatant derived from the cell lysate after ultracentrifugation was raised to 2.4 M ammonium sulfate (60% saturation). This step concentrated the RNAP I transcription apparatus in the pellet while removing small RNA species (tRNA), resulting in an extract with modest RNAP I transcriptional activity (Fig. 2, lane1). RNAP I transcriptional activity was assayed using the template pDR10, which was linearized with EcoRV, to produce a run-off transcript of 405 nucleotides (Fig. 3). This template also carries the 5 S rRNA gene, which is transcribed by RNAP III. A significant step in the protocol is the precipitation of the RNAP I transcription apparatus in low salt buffer. The residual ammonium sulfate in the resuspended 60% ammonium sulfate pellet was removed from the sample by extensive dialysis against buffer lacking salt, followed by centrifugation. Less than 25% of the total protein precipitated under these conditions. We found significant RNAP I activity in this pellet (termed ``low salt pellet''), while the RNAP III activity was found in the supernatant (Fig. 2, lanes2 and 3). Efficient solubilization of the RNAP I transcription apparatus in the pellet was achieved in 400 mM ammonium sulfate (lanes4 and 5). To prepare the sample for column chromatography, the extract was dialyzed into buffer with a conductivity equivalent to 100 mM KCl. Roughly half of the total protein in this sample was insoluble, which was removed by centrifugation. Fractionation of the crude cell lysate by precipitation in high and low salt buffers results in an RNAP I extract having more specific transcriptional activity per equivalent volume and less than 10% of the protein found in the initial crude lysate. The composition of the crude cell lysate after ultracentrifugation is very complex with numerous inhibitory compounds. The high activity of the RNAP I extract may reflect both the efficient recovery of the RNAP I apparatus and the removal of such inhibitors. Using this protocol, we have produced transcription extracts from as little as 5 g of cells to as much as several hundred g of cells. The resolution of the components of the RNAP I transcription system by column chromatography was significantly enhanced by the initial precipitation and solubilization treatments.

Figure 2: The RNAP I and III transcriptional activities of the intermediate fractions. The DNA template used in the assays, pDR10 linearized with EcoRV, contains both RNAP I and III transcription units. The transcription assay of the 10-60% ammonium sulfate cut was run in lane1; the low salt pellet, lane2; the low salt supernatant, lane3; the insoluble low salt pellet, lane4; and the solubilized low salt pellet, lane5. Equivalent portions (percentage of total volume) of each fraction were assayed.


Figure 3: The structure of the yeast rDNA-containing plasmids. Top, the rDNA repeat. The positions of the rDNA coding regions are indicated by the closedboxes, the lightlyshadedboxes are the external and internal transcribed spacer regions, the darklyshadedbox is the RNAP I promoter, and the stripedbox is the enhancer (enh). The RNAP I (35 S) and III (5 S) transcripts are represented by arrows. Bottom, the transcription template pDR10 with relevant restriction sites. The RNAP I run-off transcripts directed by EcoRV- or KpnI-linearized templates are indicated along with the transcript sizes.


This supernatant in 100 mM KCl was chromatographed on a strong anion exchange Q support using gradient elution. Transcription assays with combinations of fractions identified three well separated, distinct activities that were each required for specific RNAP I transcription (Fig. 4). In the absence of fraction A, high molecular weight radioactive nucleic acids were reproducibly produced in the transcription assay (lane2). Each of the three fractions have been subjected to further chromatography, and we have been unable to further resolve any of the three fractions into subfractions, suggesting that this transcription system has three essential components, each of which consists of one or multiple proteins that remain associated under the fractionation conditions. By analogy to other eucaryotic RNAP I transcription systems, we expect the polypeptide composition of at least two of the three components to be quite complex. In the following experiments, we examine the possible functions of each component.

Figure 4: Column chromatography of the RNAP I transcription extract on Q matrix. A, the protein and salt concentration profiles of the Q column fractions. The fractions containing the A, B, and C activities are indicated by the bars at the top. B, reconstitution of specific RNAP I transcription. Fractions A, B, and C were all present in the assay run in lane1. In each of the subsequent lanes, one of the fractions was omitted.


RNA Polymerase Activity in the Fractions

The fraction containing RNAP I was identified by nonspecific transcription assays. Transcription of a nonspecific DNA template (such as salmon sperm DNA) is a measure of the catalytic activity of RNA polymerase and is not influenced by the presence of ancillary factors. Fractions A, B, and C were each assayed for RNA polymerase activity. Only fraction B had significant RNA polymerase activity (Table 1), and it was largely resistant to alpha-amanitin, which inhibits RNAP II(34) , and tagetitoxin, which inhibits RNAP III(35) . A trace amount of RNA polymerase activity was observed in the leading edge of the C activity peak from the Q column. Fractions from the trailing edge of the C activity peak had no polymerase activity yet were able to reconstitute specific RNAP I transcription with the A and B fractions. Consistent with B containing RNAP I, an activity in this fraction is specifically deficient in stationary phase cells (see below).

Template Commitment Activity in the Fractions

Promoter recognition is an obligatory initial step in transcription. In eucaryotes, transcription factors first bind promoter sequences to form stable preinitiation complexes, which then recruit other transcription factors and RNA polymerase to the promoter. These DNA binding proteins can be detected by transcriptional competition assays between two templates that direct the synthesis of different length transcripts (see Fig. 3for template structures). The fractions, either alone or in combination, are incubated with one template while the complementing fractions are incubated with the second template (Fig. 5A). The two reactions are then mixed and incubated to permit assembly of complete complexes; then, the transcription assay is initiated. As expected, when all three fractions were preincubated together on the first template, a transcription complex was formed that was stable to a challenging template (Fig. 5B, lanes1 and 2). To determine which fraction is responsible for the initiation of complex assembly, different combinations of the three fractions were preincubated with the two templates. These experiments showed that fraction A was necessary and sufficient to catalyze the formation of the stable transcription complex (lane3). Preincubation of fraction A with fraction B did significantly increase the quantity of transcript produced, and, to a lesser extent, C had the same effect (lanes4 and 5). These experiments show that the A fraction contains the transcription factor responsible for initiating assembly of the transcription complex, presumedly by tightly binding the DNA template.

Figure 5: Template commitment assay. A, the experimental design. The templates were pDR10 linearized with either KpnI or EcoRV (templates1 and 2, respectively) and are shown in Fig. 3. The templates were incubated separately with complementary subsets of the three fractions and then mixed and allowed to incubate together. Assays were initiated by the addition of nucleotides. B, transcription assay results. The positions of the transcripts from templates 1 and 2 are indicated in the leftmargin. Above each lane is indicated which fractions were incubated with the templates during the initial incubation.


TBP Content of the Fractions

TBP, originally identified as an RNAP II transcription factor, is also an essential component of the RNAP I transcription factor SL-1 and homologues(11, 36) . Subsequently, it was shown to be a pivotal factor in all three nuclear transcription systems (see (37) for a recent review). It has been directly demonstrated that TBP is an essential component of the yeast RNAP I transcription apparatus(12, 13) . With the goal of identifying the fraction A, B, or C that contains the TBP-containing RNAP I factor, we performed Western blotting analysis of each fraction using anti-TBP antiserum. Fraction A was found to contain virtually all of the TBP, although a slight amount was also observed in the B fraction (data not shown). Next, we examined the Q column elution profiles of A activity and TBP. If TBP is a component of the A activity, we would expect to find a correlation between A activity and TBP content. All of the chromatographic fractions containing A activity also contained TBP, consistent with the suggestion that the A fraction contains the yeast SL-1 homologue. TBP eluted off the Q column in a broad peak from less than 180-250 mM KCl, while the A activity eluted in a much sharper peak from 215 to 250 mM (data not shown). The broad elution profile of TBP may be due to the different populations of TBP. The presence of multiple TBP complexes in the cell has recently been demonstrated by Poon and Weil(38) . They fractionated a whole cell extract using size exclusion chromatography and identified TBP-containing fractions using Western blotting analysis. Less than one-third of the total TBP eluted as a monomer, while the remainder was found in high molecular weight complexes.

Growth Rate Regulation of the RNAP I Transcription Complex

The strong relationship between growth rate and rRNA synthesis has been reported in a number of systems ranging from bacteria to higher eucaryotes. While the molecular mechanisms in procaryotes remain rather obscure, in eucaryotes it is clear that one mechanism of regulation of rRNA synthesis involves a modification of a factor closely associated with RNAP I. We analyzed RNAP I- and III-specific transcriptional activities in extracts prepared from cells that had either a reduced growth rate or had stopped growing altogether. At high cell densities, the cells leave exponential phase, during which energy is derived primarily through the fermentation of glucose, and enter a transition phase that is characterized by a slower growth rate and a high rate of respiration. Eventually, the cells leave the transition phase and enter stationary phase. These cells are morphologically and physiologically distinct from growing cells(39) . Small scale transcription extracts (low salt pellets and supernatants) were prepared from portions of the culture harvested during the transition and stationary phases.

The extracts prepared from the transition phase cells completely lacked specific RNAP I transcriptional activity (Fig. 6A, lane2). Extracts from stationary phase cells had a pronounced increase in the synthesis of high molecular weight RNA and reproducibly contained a trace of RNAP I-specific activity (lane3). The regulatory response in transition phase cells is specific for RNAP I, as extracts prepared from these cells retained significant RNAP III activity (5 S rRNA) (Fig. 6B, lane1). In contrast, extracts prepared from the stationary phase culture exhibited a decrease in RNAP III-specific transcription (lane2). This protocol for extract preparation minimizes the chance of inactivation due to trivial reasons as the RNAP I and III extracts are prepared from the same batch of cells carried through the same steps. Only at the final step is the specific RNAP I activity (low salt pellet) separated from the RNAP III activity (supernatant).

Figure 6: Transcriptional activities of extracts prepared from cells in various phases of growth. A, RNAP I-specific transcriptional activity was assayed in low salt pellets prepared from exponential phase cells (lane1), transition phase cells (lane2), and stationary phase cells (lane3). B, the RNAP III-specific transcriptional activity in the low salt supernatants prepared from transition phase cells (lane1) and stationary phase cells (lane2).


Using the reconstituted transcription system, we were able to determine which of the three fractions was affected by entry of the culture into transition phase. The inactive low salt pellet from transition phase cells was supplemented with the A, B, or C fractions isolated from exponential phase cells (Fig. 7). The B fraction alone was able to restore significant specific RNAP I transcriptional activity to the transition phase extract (lane3), while addition of the A and C fractions had no effect. These results are consistent with a mode of regulation analogous to that which has been observed in higher eucaryotes involving the modification of a factor associated with the RNA polymerase.

Figure 7: Restoration of specific RNAP I transcription in extracts prepared from transition phase cells. The assays in lanes1-4 all contained low salt pellets from transition phase cells, supplemented with the fractions A, B, or C as indicated. The activity in lane5 was reconstituted from the combination of the three A, B, and C fractions. The level of activity present in the B fraction alone is shown in lane6.


DISCUSSION

Here, we have presented the resolution and initial characterization of three components of the yeast RNAP I transcription apparatus. Several factors seem to be critical for the preparation of extracts containing the robust RNAP I transcriptional activity that is required for column chromatography. First, the cells must be harvested while they are still in exponential growth phase, as yeast RNAP I transcription extracts faithfully reflect the decrease in transcription observed in vivo as cells leave the exponential phase of growth(26, 27) . Additionally, when breaking cells with glass beads, we found it important to carefully monitor the process. The most active extracts were obtained when 50-75% of the cells had their walls disrupted. These cells can be distinguished using phase contrast microscopy as they appear dark, while intact cells are light and more refractive. When the cells were extensively homogenized, which could be confirmed by the lack of any large wall fragments, little or no activity was recovered. The third important consideration is the removal of extraneous proteins with the low salt precipitation. Material that had not been precipitated in the low salt buffer (for example the 10-60% ammonium sulfate cut) was much less effectively resolved into the three fractions on an anion exchange column, and the material obtained was much less active. Precipitation of the RNAP I transcription components has been previously observed in mouse cell extracts(40) . But unlike the precipitation of the yeast RNAP I and associated factors we report here, precipitation of the mouse components was dependent upon the addition of an rDNA template.

Not unexpectedly, the yeast system appears to be very similar to higher eucaryotic RNAP I systems. We have preliminary evidence for the function of the components in two of the three fractions. Based on DNA binding activity and TBP content, fraction A may contain a factor similar to SL-1 studied in other systems. Using a template commitment assay, we have shown that the A activity is responsible for initiating the assembly of the transcription complex. We have observed a protein-DNA complex formed between the RNAP I promoter and a component in the A fraction by gel retardation analysis. (^2)The relationship between this complex and RNAP I transcription remains to be determined.

The B fraction contributes RNAP I. Presently, we are further purifying the B activity from exponential phase cells. In more purified preparations having B activity, we have been able to detect two polypeptides that have a molecular weight similar to the two largest subunits of RNAP I. A factor in this fraction is also the target of growth rate regulation. Extracts prepared from slowly or non-growing cells can be rescued by exogenous fraction B. This suggests that the inactivation of the transcription complex does not occur by the accumulation of an inhibitor but rather through the inactivation of an essential component of the transcription apparatus. The relationship between the regulated factor and the RNAP I enzyme awaits further purification of the B fraction. One advantage of addressing this question in the yeast system is that unlike the RNAP I complex of other systems, the subunits of yeast RNAP I have been quite well characterized, so any alteration in subunit composition or subunit size can be traced to a specific gene product. The function of the third fraction, C, remains obscure. Although it is essential in the transcription assays, it does not appear to have a strong binding affinity for RNAP I promoter DNA. It may function by facilitating or stabilizing the binding of the fraction of the A activity to the promoter or, alternatively, it may be involved in recruiting the polymerase to the transcription complex.

FOOTNOTES

*
This work was supported by Grant GM47881 from the National Institutes of Health, Award HN2-016 from the Oklahoma Center for the Advancement of Science and Technology (OCAST), and a Junior Faculty Research Award from the University of Oklahoma. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 405-325-1683; Fax: 405-325-7619.

(^1)
The abbreviations used are: RNAP, RNA polymerase; Ubf, upstream binding factor; TBP, TATA binding protein.

(^2)
E. Clarke and J. Crowell, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. Martin Schmidt for providing purified TBP, TBP-containing E. coli extract, and TBP antisera and Jeff Goodell, Claire Chazaud, Aaron Brainard, and Kathy Dodd for expert technical assistance. We also thank the Molecular Biology Resource Facility at the University of Oklahoma Health Sciences Center for the synthesis and purification of synthetic oligonucleotides.

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