(Received for publication, November 8, 1994; and in revised form, January 11, 1995)
From the
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.
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). ()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.
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) .
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 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.
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
-amanitin concentration in the assays
was 75 µg/ml.
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
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.
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.
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.
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. ()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.