©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Inactivation of Two Components of the Multiprotein Transcription Factor TFIIIB in Cycloheximide Growth-arrested Yeast Cells (*)

Giorgio Dieci (§) , Laura Duimio , Giovanna Peracchia , Simone Ottonello (¶)

From the (1) Institute of Biochemical Sciences, University of Parma, via della Scienze, I-43100 Parma, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.


INTRODUCTION

The intracellular levels of the RNA components of the eukaryotic translational apparatus are regulated in response to growth conditions, and nuclear run-on analyses have shown that this regulation occurs in large part at the transcriptional level (1, 2 and references therein).

Both in the yeast Saccharomyces cerevisiae and in higher eukaryotes, rRNA synthesis by RNA polymerase (pol)() I is known to be down-regulated by a number of treatments, including growth arrest induced by protein synthesis inhibitors, starvation for an essential nutrient, and growth into stationary phase (1-4 and references therein). Interestingly, extracts prepared from growth-restricted mammalian cells have an impaired capacity for RNA pol I transcription due to depletion or modification of an essential transcription factor (1, 5) .

5 S RNA and tRNA synthesis by RNA pol III is similarly reduced following a cycloheximide-induced growth arrest, under conditions in which RNA pol II transcription is only slightly affected (1) . A negative biosynthetic response to cycloheximide administration has been observed both in mammalian cells (1) and, albeit to variable extents, also in yeast (3, 4, 6) . Soluble extracts derived from mouse cells, either treated with cycloheximide or grown into stationary phase, mimic the in vivo response and down-regulation of RNA pol III transcription in these cells has been shown to result mainly from a reduced activity of the general class III transcription factor TFIIIB (7). The same transcription factor has been found to be involved in the down-regulation of class III gene transcription that occurs in differentiating mouse F9 cells and in frog oocytes undergoing mitosis (8, 9). These findings thus raise the possibility that TFIIIB may represent a general target for the negative modulation of RNA pol III transcription in eukaryotic cells.

In view of the recent discovery that TFIIIB from various organisms is a multiprotein transcription factor (reviewed in Ref. 10), a further question of interest concerns the identification of the TFIIIB component(s) that is either modified or destabilized under protein synthesis inhibitory conditions. The highest resolution of the TFIIIB complex has been achieved, so far, for the transcription factor from yeast. In this system, two distinct polypeptides, TFIIIB70 (also known as BRF, TDS4, or PCF4) and the TATA box-binding protein (TBP), plus a fraction (B") that is enriched in a 90-kDa polypeptide have been shown to be necessary and sufficient to reconstitute TFIIIB activity (11-17). A fourth component, TFIIIE, which is also required to reconstitute in vitro class III transcription in the presence of a partially purified TFIIIB fraction, has recently been resolved in our laboratory and found to co-fractionate with highly purified B" fractions (18) . Notwithstanding the availability of cloned or highly resolved class III transcription components, tRNA and 5 S RNA synthesis have not yet been analyzed in crude extracts of cycloheximide-treated yeast cells, and nothing is known about the molecular events underlying negative modulation of RNA pol III transcription under conditions of protein synthesis inhibition.

We are interested in defining the targets and the mechanisms of inactivation of yeast pol III transcription in response to cycloheximide-induced protein synthesis inhibition. We were also intrigued by the possibility that TFIIIE might be involved in such response. The effect of cycloheximide upon tRNA and 5 S RNA synthesis has thus been initially re-examined under in vivo pulse labeling conditions. We have found that the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of 20 and 10 min, respectively, in cycloheximide-treated cells and that in vitro transcription of a tRNA gene by extracts of treated cells faithfully mimics the in vivo response. Only TFIIIB activity is severely impaired in these extracts, and a selective loss of TFIIIB70 and B" activities, but not TBP activity, fully accounts for the observed inactivation.


EXPERIMENTAL PROCEDURES

Cell Growth and Pulse Labeling Analysis

The diploid, protease-deficient S. cerevisiae strain RH804C775D ( lys2 leu2 pep4-3/a leu2 trp1::URA3 gal2 pep 4-3, a gift of H. Riezman, University of Basel, Switzerland) was used throughout this work. Cells were grown at 30 °C in phosphate-depleted YEPD medium (19). Cycloheximide (Fluka, 10 µg/ml) was added to yeast cultures grown to a density of 10 cells/ml; mock-treated aliquots of the same cultures were used as controls. For pulse labeling experiments (20) , aliquots of either cycloheximide-treated or control cultures (a total of 10 cells each) were collected by centrifugation at various times after cycloheximide addition and resuspended in 0.5 ml of the corresponding growth medium. Cells were then incubated for 10 min in the presence of radioactive [P]orthophosphate (20-30 µCi, 8500 Ci/mmol), washed twice with ice-cold growth medium, and frozen in liquid nitrogen in preparation for RNA extraction. To determine the time course of radioactive phosphate accumulation by either control or cycloheximide-treated cells, samples were taken at 10-min intervals from a scaled-up incubation mixture, washed twice with ice-cold growth medium, and counted.

RNA Isolation and Analysis

Total RNA was extracted with the hot phenol procedure (21) . After the addition of ammonium acetate (3.75 M, final concentration) and ethanol (2.5 volumes), samples were kept on dry ice for 20 min, and RNA was collected by centrifuging at 12,000 g for 30 min. The pellets were then washed once with 70% ethanol, dissolved in diethyl pyrocarbonate-treated water, and used for either spectrophotometric, electrophoretic, or chromatographic analyses. RNA samples (15 µg of total RNA) were fractionated on 6% polyacrylamide, 7 M urea gels. The gels were first stained with ethidium bromide to monitor total amounts of the different RNA species; they were subsequently exposed overnight for autoradiography, followed by the quantitation of individual RNA bands by Cerenkov counting. Poly(A) RNA was resolved by chromatography on oligo(dT)-cellulose (Boehringer) following the manufacturer's instructions.

Plasmid DNAs

Plasmids pUCLeu3, containing a yeast tRNA gene, pPM16, containing a yeast tRNA gene, and pUC9-5S, containing the yeast 5 S rRNA gene (from P. A. Weil, Vanderbilt University School of Medicine, Nashville, TN), were used as templates for in vitro transcription reactions. Plasmids pET3b(rTFIIDY) (22) and pSH360 (12) were used to express yeast TBP and TFIIIB70 in Escherichia coli and were kindly provided by J. M. Egly (INSERM, Strasbourg, France) and S. Hahn (Fred Hutchinson Cancer Research Center, Seattle, WA). Plasmid pETIIIA (23) was used for the expression of yeast TFIIIA.

Extract and Transcription Factor Preparations

Nuclear extracts were prepared from either control or cycloheximide-treated cells grown to a final density of 1-1.5 10 cells/ml (18). Ammonium sulfate concentrations ranging from 0.5 to 0.9 M were tested for their nuclear extraction ability. The resulting extracts were found to exhibit essentially identical in vitro transcriptional activities (see below), and a final salt concentration of 0.65 M was routinely used for nuclear extract preparation. Transcription factor fractions (TFIIIB, TFIIIC, and TFIIIE) and polymerase were partially purified from control extracts as described previously (18) . Fraction B" was extracted from chromatin pellets and partially purified on BioRex 70 (Bio-Rad) according to Kassavetis et al.(17) . Recombinant yeast TFIIIA was expressed in E. coli and purified to near homogeneity as described previously (23) . Recombinant yeast TBP and TFIIIB70 were purified from E. coli-soluble lysates using modifications of previously described procedures (12, 22) . After an initial fractionation on DEAE-Sephacel and heparin-Ultrogel (22) , rTBP was purified to near homogeneity by gel filtration on Ultrogel AcA-54 (IBF, 2 150 cm column) equilibrated, and eluted with 25 mM Tris-HCl (pH 8.0), 0.2 mM EDTA and 0.5 M KCl at a flow rate of 0.35 ml/min. Histidine-tagged rTFIIIB70 was purified to about 50% homogeneity by chromatography on Ni-NTA agarose (Quiagen) under native conditions, using 250 mM imidazole dissolved in 50 mM sodium phosphate (pH 8.0), 0.3 M NaCl, 1.5 mM -mercaptoethanol, plus protease inhibitors (1 µM each of leupeptin and pepstatin, 0.1 mM phenylmethylsulfonyl fluoride, and 0.5 mM benzamidine) as eluent. The in vitro complementation of extracts derived from TBP mutant yeast cells (YSB67 strain, 24) and the transcriptional stimulation of extracts prepared from wild type haploid cells (12) were initially used to assay for rTBP and rTFIIIB70, respectively. Protein concentrations were determined by the method of Bradford (25) , using bovine serum albumin as standard.

Transcription Assays

Standard transcription assay mixtures contained 80 fmol of template DNA, 15 mM Tris-HCl (pH 7.9), 120 mM KCl, 6 mM MgCl, 10% (v/v) glycerol, 600 µM each of ATP, GTP, and CTP, 25 µM UTP, and 5-10 µCi of [-P]UTP (800 Ci/mmol). Transcription assays were carried out in a final volume of 20 or 37 µl, in the case of either crude extracts or variously reconstituted transcription systems. The amounts of extracts, fractions, and purified components employed for each experiment are indicated in the figure legends. Multiple round transcription reactions were incubated for 20-30 min at room temperature. The concentration of transcriptionally active components in either partially purified transcription factor fractions and in recombinant transcription factor preparations was determined by single round transcription assays as described previously (18, 26) . Reactions were terminated by the addition of 10 µl of a solution containing 4% (v/v) SDS and 5 mg/ml of carrier RNA (Sigma, type R-6625 T6). After phenol-chloroform extraction and ethanol precipitation, RNA products were analyzed by electrophoresis on 10% polyacrylamide, 8 M urea gels as described previously (18) . Transcription products were visualized by autoradiography of wet gels and were quantitated by measuring Cerenkov radiation in gel slices.

Western Blot Analysis

Equal amounts of total protein from either extracts of cycloheximide-treated cells or from the corresponding control extracts were electrophoresed, along with molecular weight markers, on polyacrylamide-SDS gels (27) . Electrotransfer to nitrocellulose membranes (Schleicher & Schuell, BA-85) and immunoblotting were carried out as described by Harlow and Lane (28) . TFIIIB70 was detected with an antiserum raised against the partially purified recombinant yeast protein (kindly provided by F. Pugh, Pennsylvania State University, University Park, PA). Polyclonal antibodies against yeast TBP were a gift of D. Hawley, University of Oregon, Eugene, OR. Horseradish peroxidase-conjugated anti-rabbit Ig antibodies and enhanced chemiluminescence reagents (Amersham) were used according to the manufacturer's instructions to detect antigene-bound primary antibodies.


RESULTS

Effect of Cycloheximide upon tRNA and 5 S RNA Synthesis in Yeast Cells

Before analyzing RNA pol III transcription in cell-free extracts of cycloheximide-treated cells, we re-examined the effect of this protein synthesis inhibitor on tRNA and 5 S RNA synthesis under in vivo pulse-labeling conditions. To this end, exponentially growing yeast cells were treated for various periods of time with cycloheximide, at a concentration (10 µg/ml) which reversibly blocked both protein synthesis and cell growth within a few minutes from its addition. Following pulse labeling of control and cycloheximide-treated cells with [P]orthophosphate (10 min), radioactively labeled RNA species were resolved by either polyacrylamide gel electrophoresis or oligo(dT)-cellulose fractionation. As shown in Fig. 1 (panels A and B), radioactive phosphate incorporation into 5 S RNA and tRNAs decreased with apparent half-times of about 10 and 20 min upon cycloheximide administration, and it leveled off at 10 and 15% of the corresponding control values after about 2 h. Under identical experimental conditions, labeling of total poly(A) and poly(A) RNA, as estimated from the incorporation of radioactive phosphate into RNA species that either bound to or flowed through oligo(dT)-cellulose, diminished with apparent half-times of 90 and 5 min, respectively (data not shown). Consistent with the fast decline of poly(A) RNA synthesis, labeling of the processed 5.8 S rRNA species was also rapidly inhibited in growth-arrested cells (Fig. 1A). The observation that the in vivo pulse labeling of distinct classes of RNAs decreases in growth-arrested cells with considerably different kinetics, together with the fact that radioactive phosphate accumulation proceeded linearly for at least 30 min after cycloheximide addition (data not shown), indicates that the effect of cycloheximide does not merely result from a reduced specific activity of the intracellular nucleotide pool. Moreover, no decrease in the steady state amounts of either 5 S RNA or tRNAs, as estimated from the intensity of ethidium bromide staining of the same gel used for pulse labeling analysis, was detected after 3 h of cycloheximide treatment (Fig. 1C). The effect of cycloheximide on 5 S RNA and tRNA accumulation thus occurs primarily at the level of transcription.


Figure 1: Analysis of newly synthesized and steady state levels of tRNAs and 5 S RNA in cycloheximide growth-arrested yeast cells. A, in vivo pulse labeling analysis. Cycloheximide (Cy, 10 µg/ml) was added to exponentially growing yeast cells at time 0. At the indicated times, aliquots of the culture were removed and pulse labeled with [P]orthophosphate (10 min). Total RNA was extracted and fractionated on 6% polyacrylamide, 7 M urea gels (see ``Experimental Procedures'' for details). The autoradiographic result of a representative pulse labeling experiment is shown; the migration positions of 5.8 S rRNA, 5 S rRNA, and tRNAs are indicated. B, quantitation of radioactive, newly synthesized tRNA () and 5 S RNA () transcripts at different times after cycloheximide addition. Gel slices corresponding to tRNA, 5 S rRNA, and background areas were excised and, after background deduction, radioactivity associated to each RNA species was determined; data are the average (± S.D.) of three independent experiments. C, ethidium bromide staining of the same gel shown in panel A.



Extracts of Cycloheximide Growth-arrested Cells Have an Impaired Capacity for RNA Polymerase III Transcription

To begin to elucidate the molecular mechanisms responsible for the down-regulation of pol III transcription in cycloheximide growth-arrested cells, extracts were prepared from cell cultures that had been exposed to the inhibitor for either 1.5 or 3 h under the above described experimental conditions, as well as from parallel cultures of mock-treated cells. Extracts were prepared from the same number of cells and contained nearly identical amounts of total protein. Parallel transcription assays were carried out with increasing amounts of either extract, using the yeast tRNA gene and the 5 S rRNA gene as templates. As shown in Fig. 2A, extracts of cells harvested at different times from cycloheximide addition became increasingly less competent for tRNA gene transcription with a 70% inactivation after 1.5 h and almost no detectable activity after 3 h. A different in vitro response was observed in the case of 5 S RNA synthesis, with practically no variation of transcriptional activity after 1.5 h of treatment and a nearly complete loss of transcription after 3 h. Extracts derived from treated cells thus appear to mimic cycloheximide-induced down-regulation of pol III transcription. However, at variance with tRNA gene in vitro transcription, which faithfully reproduces the in vivo response (cf. Fig. 1B and 2A), the time course of inactivation of in vitro 5 S RNA transcription was found to be slower than that measured in intact cells (cf. Fig. 1B and 2B).


Figure 2: Comparison of the transcriptional capacities of control extracts and extracts derived from cycloheximide-treated cells. A, tRNA gene transcription. Increasing amounts of extracts prepared from cells that had been exposed to cycloheximide (Cy) for either 1.5 h (lanes 4-6) or 3 h (lanes 10-12), and the corresponding control extracts (Co, lanes 1-3 and 7-9, respectively) were tested for their ability to transcribe the yeast tRNA gene. The following amounts of extract protein were added to individual reaction mixtures: 8 µg, lanes 1, 4, 7, and 10; 16 µg, lanes 2, 5, 8, and 11; 32 µg, lanes 3, 6, 9, and 12. B, 5 S rRNA gene transcription. Lanes are numbered as in panel A; the same experimental conditions were used. The positions of transcripts (tRNA, 5 S RNA) after resolution on polyacrylamide gels are shown.



As verified by experiments in which a control extract was preincubated with cycloheximide prior to transcription, the impaired transcriptional capacity of extracts prepared from treated cells is not due to a direct effect of the inhibitor on the transcription machinery (data not shown). Furthermore, extracts of cycloheximide-treated cells did not inhibit control extracts when the two were mixed together prior to in vitro transcription (Fig. 3). This result indicates that the transcriptional impairment of extracts derived from treated cells is not due to the presence of a diffusible inhibitor and that these extracts do not trans-inactivate any transcriptional component of the control extract. Rather, it argues that inactive extracts have reduced amounts of a positive activity that is required for tRNA and 5 S RNA transcription.


Figure 3: In vitro transcription in mixed extracts. Transcription reaction mixtures, programmed with the yeast tRNA gene, contained either 8 µg of control extract (Co, lane 1), 8 µg of extract derived from cells that had been exposed to cycloheximide for 3 h (Cy, lane 2), or 8 µg of each type of extract (lane 3). The position of transcripts (tRNA) after resolution on a polyacrylamide gel is shown.



TFIIIB Activity Is Selectively Impaired in Extracts of Cycloheximide-treated Cells

To identify the class III component(s) which is responsible for the cycloheximide-induced down-regulation of pol III transcription, we initially tried to restore tRNA gene transcription by extracts of treated cells with the addition of individual transcription factors partially purified from control cells. As shown in Fig. 4, supplementation of either TFIIIC, TFIIIE, or polymerase to extracts of treated cells had no effect on transcription (cf.lane 2 with lanes 6-8). In contrast, the addition of partially purified TFIIIB fully restored transcription (lanes 3-5). Similar experiments with the 5 S rRNA gene showed that TFIIIB also restores 5 S RNA transcription (data not shown). TFIIIB thus appears to be primarily responsible for the impairment of tRNA synthesis that occurs in cycloheximide-treated cells, and a reduced activity of this component also accounts for the delayed decline of in vitro 5 S RNA transcription.


Figure 4: Complementation of extracts derived from cycloheximide-treated cells by the TFIIIB fraction. The transcriptional activity of an extract prepared from treated cells (Cy, 3 h of exposure to cycloheximide, 16 µg of total protein) was measured in the absence of additional factors (lane 2), or in the presence of partially purified TFIIIB (B, 0.5, 1, and 2 µg of total protein, lanes 3-5), TFIIIC (C, 0.6 µg, lane 6), polymerase (p, 0.4 µg, lane 7), or TFIIIE (E, 0.5 µg, lane 8). The transcriptional output of the control extract (Co, 16 µg of total protein) is shown for comparison in lane 1.



An intrinsic restraint of ``add back'' experiments is their inability to detect even significant variations of components other than the one that is limiting for transcription. We thus assayed the activity of individual class III transcription components by supplying graded amounts of extracts from either control or cycloheximide-treated cells to partially reconstituted transcription systems providing an excess of all components except for the one being tested. As shown in , TFIIIB activity is about 10-fold reduced in extracts of treated cells, whereas nearly identical TFIIIA, TFIIIC, TFIIIE,and polymerase activities are present in either type of extract.

While confirming the results of the add back experiment as to the primary involvement of TFIIIB in the transcriptional response to cycloheximide, the latter data show that the reduced activity of this component parallels the decrease of total transcription capacity measured in unfractionated extracts. tRNA and 5 S rRNA transcription by control extracts is limited by different activities, either TFIIIB or TFIIIA, yet no transcriptional component other than TFIIIB has been found to vary between control extracts and extracts of treated cells. This selectivity of inactivation also indicates that the effect of cycloheximide does not merely result from a generalized alteration of the nuclear extractability of class III transcription components. Indeed, practically identical results as to the impaired transcriptional capacity of extracts derived from treated cells were obtained when varying the ammonium sulfate concentration used for nuclei extraction from 0.5 to 0.9 M (data not shown).

The TFIIIB70 and B" Components of TFIIIB Are Selectively Inactivated Following Cycloheximide-induced Protein Synthesis Inhibition

Yeast TFIIIB is a multiprotein complex constituted by at least three distinct components (reviewed in Ref. 29). To determine which of these components is negatively modulated in cycloheximide-treated cells, we first set up a reconstituted transcription system in which TFIIIB is replaced by recombinant TFIIIB70 and TBP, plus a fraction, B", that was partially purified from yeast nuclear pellets (Fig. 5A, lanes 3-9). The latter fraction was found to contain sizeable amounts of cross-contaminating TFIIIE (data not shown). At variance with its transcriptional requirement in the presence of the native TFIIIB complex (Fig. 5A, lanes 1 and 2), TFIIIE thus turned out to be dispensable for transcription in the presence of fraction B" (Fig. 5A, lanes 3 and 6). On the contrary, TFIIIB70, TBP, and B" were all required to reconstitute TFIIIB activity, and the level of tRNA transcription efficiency achieved with internally calibrated amounts of these three components was comparable to that obtained in the presence of native TFIIIB and TFIIIE (Fig. 5A).


Figure 5: Restoration of tRNA gene transcription by resolved TFIIIB subcomponents. A, reconstitution of TFIIIB activity by TFIIIB70, TBP, and B". Transcription reactions were carried out in the presence of TFIIIC (C, 0.6 µg of total protein), TFIIIE (E, 0.5 µg), and polymerase (pol, 0.4 µg), plus either the native TFIIIB fraction (0.5 µg, lane 1) or internally calibrated amounts of the three TFIIIB subcomponents (rTFIIIB70 80 ng, rTBP 40 ng, fraction B" 0.5 µg, lane 3). As indicated above each lane, all of these components were individually omitted from the latter reaction mixture (lanes 4-9), whereas only TFIIIE was omitted from reactions supported by the native TFIIIB fraction (lane 2). All the lanes shown come from the same exposure of a single gel. B, complementation of extracts derived from cycloheximide-treated cells by TFIIIB70 plus B". Transcription reaction mixtures contained 16 µg of an extract prepared from treated cells (Cy, 3 h of treatment, lanes 2-12), either alone (lane 2) or supplemented with various combinations of TFIIIB subcomponents as indicated above each lane (lanes 3-11). The native TFIIIB fraction (nat. IIIB, 0.5 µg of total protein) was added to the reaction in lane 12. A control extract prepared from mock-treated cells (Co, 16 µg of total protein) was used for the reaction in lane 1. The amounts of TFIIIB subcomponents added to the cycloheximide extract were as follows: rTBP, 40 ng; rTFIIIB70, 80 ng; B", 0.3, 0.7, 2 µg in lanes 6-8, respectively, and 2 µg in lanes 9-11. The position of transcripts (tRNA) after resolution on a polyacrylamide gel is shown.



As shown in Fig. 5B, neither TBP, B", nor a combination of these two components could restore the activity of an extract prepared from cycloheximide-treated cells. Only a slight stimulation of transcription was observed with either TFIIIB70 or TFIIIB70 plus TBP (cf. lane 2 with lanes 4 and 5), and essentially identical results were obtained upon the additional supplementation of TFIIIE (data not shown). In contrast, the same extract was restored to about 90% of the control activity by the addition of TFIIIB70 and B" (cf.lanes 1 and 9), with only a little additional recovery upon the further supplementation of TBP (lane 11). TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the cycloheximide-induced inactivation of TFIIIB. This conclusion was further supported by titration experiments, which showed that largely reduced (7-10-fold) B" and TFIIIB70 activities, but essentially unchanged levels of TBP activity, are present in extracts of treated cells (data not shown).

The observed inactivation of TFIIIB70 and B" may result from their selective destabilization under conditions of protein synthesis inhibition. Alternatively, it may derive from a cycloheximide-induced modification of these components, leading to a substantial impairment of their transcription factor activity. To begin to distinguish between these two possibilities, we used anti-TFIIIB70 antibodies for a Western immunoblot analysis of control and cycloheximide extracts. TBP was used as an internal reference for this analysis and, in agreement with the results of previous titration experiments, it was found not to vary between the two types of extract (Fig. 6A). In contrast, under identical experimental conditions, a substantial depletion of the TFIIIB70 polypeptide was detected in extracts derived from cycloheximide-treated cells (Fig. 6B).


Figure 6: Comparative analysis of the levels of TBP and TFIIIB70 in extracts derived from cycloheximide and mock-treated cells. A, TBP: 10 µg of either a control extract (Co, lane 1) or an extract prepared from cycloheximide-treated cells (Cy, 3 h of treatment, lane 2), and 30 ng of recombinant yeast TBP (rTBP, lane 3) were subjected to SDS-polyacrylamide gel electrophoresis and Western immunoblot analysis as described under ``Experimental Procedures.'' The migration position of the 27-kDa TBP polypeptide is indicated. B, TFIIIB70: identical amounts of the same extracts used for the experiment reported in panel A were analyzed. 10 ng of recombinant yeast TFIIIB70 (rIIIB) were loaded in lane 3; the migration position of the TFIIIB70 polypeptide (70 kDa) is indicated.




DISCUSSION

We have found that yeast pol III transcription, though appreciably less sensitive to cycloheximide-induced inactivation than pol I transcription, is similarly down-regulated under conditions of protein synthesis inhibition. The results of our in vivo pulse labeling analysis are in keeping with the previously reported differential sensitivity of these two transcription systems to cycloheximide (1, 3, 4, 6) . In particular, tRNA transcription declines under in vivo conditions with an apparent half-time about 4-fold longer than the corresponding half-time for pol I transcription. In turn, pol III transcription has been found to decline 4.5-9-fold more rapidly than pol II transcription, indicating that the effect of cycloheximide is not due to a generalized destabilization of the RNA biosynthetic machinery. Furthermore, the steady state levels of tRNAs and 5 S RNA do not change following cycloheximide addition, as if their turnover rates were considerably slowed-down under growth-arrested conditions. A similar divergence between the rate of synthesis and the steady state level of various class III transcripts has been observed in other systems, and the regulation of tRNA half-life has previously been reported (10, 30, and references therein). By enabling cells to maintain a level of essential pol III products that is sufficient to support basal cellular functions, this mechanism would explain the complete reversibility of the cycloheximide-induced growth arrest as observed under our experimental conditions.

Extracts of cycloheximide-treated cells programmed with a cloned tRNA gene mimic the in vivo response to protein synthesis inhibition. The same extracts did not faithfully reproduce the earlier decline of 5 S RNA synthesis that was observed under in vivo labeling conditions. Since TFIIIA has the ability to bind both the 5 S gene and the 5 S RNA transcript (10) , this discordance of behavior may result, for example, from an in vivo shortage of transcriptionally competent TFIIIA, due to a reduced availability of the ribosomal 5 S RNA binding protein YL3 in cycloheximide growth-arrested cells (20, 31) . Alternatively, it may reflect a predominant chromatin effect under in vivo conditions, arising from the peculiar chromosomal location of yeast 5 S rRNA genes within the ``spacer'' regions interposed between individual rDNA transcription units (32) . Our present in vitro data do not allow distinguishing between these two possibilities, and the only transcription component that has been found to be affected by cycloheximide treatment is TFIIIB. However, the observation that the level of TFIIIA activity is initially lower than the level of TFIIIB (), at least accounts for the delayed involvement of TFIIIB in the in vitro detected inactivation of 5 S RNA transcription.

Similar to what has previously been found in the case of growth-restricted mouse cells (7) , TFIIIB is the only class III transcription component whose activity is severely impaired in extracts of cycloheximide-treated yeast cells. The observation that an identical class III component mediates a negative transcriptional response in so diverse organisms supports the view that TFIIIB may indeed be a general target for eukaryotic pol III down-regulation. The selective impairment of TFIIIB activity under protein synthesis inhibitory conditions is consistent with the key role that this component is known to play in transcription initiation (33) . In various mammalian systems, TFIIIC has previously been shown to be implicated in both positive and negative pol III regulatory events (reviewed in Ref. 10). At variance with TFIIIC, however, TFIIIB inactivation has the potential ability not only to prevent transcription complex formation, but also to abolish initiation from preassembled transcription complexes. Particularly interesting, in this regard, is the involvement of TFIIIB in controlling the differential expression of constitutive and silk gland-specific tRNA genes in silkworms (34) , and in the down-regulation of pol III transcription during differentiation of mouse F9 cells (8) . TFIIIB is also involved in the repression of class III gene transcription that occurs in Xenopus oocytes as they go through mitosis (9) . The latter event has been found to be mediated by the phosphorylation of a TFIIIB subunit (9) , but the specific TFIIIB components that are involved in the above mentioned negative modulations are not yet known.

TFIIIB has recently been shown also to mediate an up-regulation event that is induced by phorbol esters in Drosophila Schneider S2 cells (35) . In this case, stimulation of pol III transcription correlates with an increased level of TBP, but no other TFIIIB component has been examined.

Based on the high resolution that has recently been achieved for the yeast TFIIIB complex (reviewed in Ref. 29), our studies represent the first analysis of the negative modulation of individual components of a multiprotein class III transcription complex, under conditions that determine an in vivo detectable inactivation of transcription. Two TFIIIB components, TFIIIB70 and B", are the specific targets of the pol III transcriptional response to protein synthesis inhibition. We do not yet know whether a qualitative or a quantitative variation is responsible for the observed inactivation of B", but the latter possibility appears to hold in the case of TFIIIB70. In fact, Western immunoblot analysis has revealed a substantial depletion of the TFIIIB70 polypeptide in extracts of cycloheximide growth-arrested cells. In the same extracts, TBP remains essentially constant, and it is capable of interacting with exogenously added TFIIIB70 and B" to reconstitute control levels of in vitro transcription activity. Since TBP is known to direct transcription by all three eukaryotic RNA polymerases (10, 14, 15, 24, 36) , its marked stability under protein synthesis inhibitory conditions is by itself quite remarkable. It is consistent both with the differential cycloheximide sensitivity of the three transcriptional machineries evidenced by our in vivo pulse labeling analysis, and with the fact that a component other than TBP has been found to be involved in the down-regulation of mouse RNA pol I transcription under protein synthesis inhibitory conditions (5) .

The inactivation of TFIIIB70 appears to be even more relevant if one considers that this particular TFIIIB component has previously been shown to limit pol III transcription in intact yeast cells (13) . In addition, both genetic and biochemical data have demonstrated the existence of a fairly stable interaction between TFIIIB70 and TBP (11, 12, 37, 38) . Interestingly, a computer sequence analysis of these two TFIIIB components detects the presence of a degradation promoting signal (``PEST,'' 39) in TFIIIB70, but not in TBP.() The trans-recognition and targeted proteolysis of specific components of multiprotein complexes has recently been reported (reviewed in Ref. 40). It will thus be important to establish whether similar mechanisms also pertain to the selective inactivation of the TFIIIB70 and B" components of yeast TFIIIB.

  
Table: Transcription factor activities in extracts of control and cycloheximide-treated cells



FOOTNOTES

*
This work was supported by the National Research Council of Italy (Target Project on Biotechnology and Bioinstrumentation) and by the Ministry of University and Scientific and Technological Research. 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.

§
Present address: Service de Biochimie et Génétique Moléculaire, CEA-Saclay, F91191 Gif sur Yvette Cedex, France.

To whom correspondence should be addressed. Tel.: +39-521-905646; Fax: +39-521-905151.

The abbreviations used are: pol, polymerase; TF, transcription factor; TBP, TATA box-binding protein; r, recombinant.

S. Ottonello and R. Percudani, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Gian Luigi Rossi for support and encouragement. We thank George Kassavetis for detailed instructions on the preparation of fraction B"; Howard Riezman and Steve Buratowski for yeast strains; Tony Weil, Jean Marc Egly, and Steve Hahn for plasmids; and Diane Hawley and Frank Pugh for antibodies. The assistance of Silvia Pizzi and Andrea Ballabeni in the preparation of rTBP and fraction B" is also gratefully acknowledged.


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