extfieldsofstudy: Biology Medicine

text:
Glutamine Deprivation Induces the Expression ofGADD45 and GADD153 Primarily by mRNA Stabilization*

The expression of the growtharrest- and DNA damage-inducible genes, GADD45 and GADD153/CHOP(C/EBP-homologous protein), as well as GRP78 (glucose-regulatedprotein of 78 kDa) was examined in several human breast cell lines subjected to acute glutamine (GLN) deprivation. GLN deprivation caused rapid elevation of GADD45 andGADD153/CHOP mRNA levels in cells that were highly dependent upon GLN for growth and viability. BothGADD mRNAs were rapidly elevated up to several hundred-fold. In contrast, GRP78 expression was elevated by no more than 4-fold by GLN deprivation. The magnitude ofGADD up-regulation roughly correlated with the extent of GLN dependence of each cell line. The levels of all three mRNAs were responsive to alterations of ambient GLN content in a physiologically relevant concentration range that corresponded to the affinities of cellular GLN transporters. Provision of GLN-derived metabolites partially inhibited the induction of GADDexpression in GLN-deprived cells. Nuclear run-on assays and mRNA decay studies suggested that the primary mechanism leading to increasedGADD mRNA levels was not transcriptional, but rather that GADD45 and GADD153/CHOPexpression were up-regulated in response to GLN deprivation via marked stabilization of these mRNAs. These results suggest that the expression of GADD genes contributes to growth arrest and/or protection from metabolic damage during GLN-poor conditions.

The expression of the growth arrest-and DNA damage-inducible genes, GADD45 and GADD153/CHOP (C/ EBP-homologous protein), as well as GRP78 (glucoseregulated protein of 78 kDa) was examined in several human breast cell lines subjected to acute glutamine (GLN) deprivation. GLN deprivation caused rapid elevation of GADD45 and GADD153/CHOP mRNA levels in cells that were highly dependent upon GLN for growth and viability. Both GADD mRNAs were rapidly elevated up to several hundred-fold. In contrast, GRP78 expression was elevated by no more than 4-fold by GLN deprivation. The magnitude of GADD up-regulation roughly correlated with the extent of GLN dependence of each cell line. The levels of all three mRNAs were responsive to alterations of ambient GLN content in a physiologically relevant concentration range that corresponded to the affinities of cellular GLN transporters. Provision of GLN-derived metabolites partially inhibited the induction of GADD expression in GLN-deprived cells. Nuclear run-on assays and mRNA decay studies suggested that the primary mechanism leading to increased GADD mRNA levels was not transcriptional, but rather that GADD45 and GADD153/CHOP expression were up-regulated in response to GLN deprivation via marked stabilization of these mRNAs. These results suggest that the expression of GADD genes contributes to growth arrest and/or protection from metabolic damage during GLNpoor conditions. GADD45 (growth arrest and DNA damage-inducible gene) is a p53-responsive gene encoding a protein that interacts with p21/WAF1/CIP1 as well as proliferating cell nuclear antigen (1)(2)(3). GADD153 encodes a C/EBP-homologous protein that binds to C/EBP isoforms, and these heterodimers bind novel DNA recognition sites (4,5). Expression of GADD genes is induced by medium depletion and by genotoxic agents that cause DNA damage (6 -9). Exogenous expression of GADD45 or GADD153 can cause antiproliferative effects (4, 10 -12). GADD45 may inhibit cell proliferation by a mechanism involving its association with p21/WAF1/CIP1 (1,3). Expression of both GADD45 and p21/WAF1/CIP1 are up-regulated by wildtype p53, and these proteins may play a causal role in the mechanism of growth repression by this tumor suppressor (2,(13)(14)(15). GADD153 expression is induced by genotoxins and inhibitors that block protein glycosylation and/or induce stress in the endoplasmic reticulum (ER) 1 and may play a causal role in the induction of cell death following ER stress (16). Expression of exogenous GADD153 causes apoptosis in myoblastic leukemia cells (12), and endogeneous GADD153 expression has recently been linked to both leukemia cell apoptosis following etoposide treatment (17) and renal apoptosis following tumicamycin treatment (18).
GRP78 (glucose-regulated protein of 78 kDa or BiP) is a molecular chaperonin that aids in the synthesis and folding of glycoproteins in the ER (19). GRP78 expression is induced by glucose deprivation or any treatment that causes the accumulation of unfolded or malfolded proteins in the ER (20,21). In fact, many treatments that lead to GRP78 expression (inhibition of protein glycosylation, glucose deprivation, and hypoxia) also induce GADD153 expression (16,22,23).
Recently, it was shown that essential amino acid deprivation induced GADD153 expression by both transcriptional and posttranscriptional mechanisms (24). In addition, GADD153 promoter activity was induced by starvation for both essential and nonessential amino acids (24). In contrast, little is known of what effects nutrient availability has on GADD45 expression. The amino acid GLN is a key regulator of cellular proliferation and survival, but there is no information regarding the mechanism by which GLN exerts this effect. In an attempt to gain insight into this phenomenon, we examined the expression of GADD45, GADD153, and GRP78 in subconfluent cultures of human breast cell lines subjected to acute GLN deprivation. The data presented here demonstrate that both GADD153 and GADD45 expression is controlled by GLN availability primarily through mRNA stabilization. Thus, GLN elicits a distinct molecular response, destabilization of GADD mRNAs. This response was particularly pronounced for GADD45 mRNA. Moreover, in a series of human breast cell lines, there is a direct correlation between the response of GADD mRNA expression to GLN deprivation and the GLN dependence of proliferation. The results suggest that the antiproliferative response to GLN starvation may involve induction of GADD45 and/or GADD153 expression by a mechanism that includes stabilization of this mRNA. This represents the first report of a potential molecular mechanism for the antiproliferative effects of glutamine deprivation. cals were obtained from Sigma or Fisher. HBL100, T47D, MB175, HS578Bst, SKBR3, and BT483 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). An additional description of these cell lines was published previously (25). TSE cells were established from a primary ductal breast carcinoma and kindly provided by Dr. Simon Powell (Massachusetts General Hospital, Radiation Oncology, Boston, MA). TSE.06 cells were obtained by adaptation of TSE cells to low GLN conditions. Tissue culture plasticware was purchased from Falcon (Becton Dickinson Labware, Franklin Lakes, NJ), except for 24-well tissue culture plates, which were purchased from Costar Corp. (Cambridge, MA). CulturPlates™, UniFilter™ plates, and the TopCount microplate scintillation counter were obtained from Packard Instrument Company (Meriden, CT). L-Glutamic acid assay kits were purchased from Roche Molecular Biochemicals.
Cell Culture-Human cell lines were grown in 75-cm 2 T-flasks at 37°C under a humidified atmosphere of 5% CO 2 , 95% air. All of the cells, except TSE.06, were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4 mM L-GLN, 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin B, and 10 g/ml bovine insulin (complete DMEM). To obtain TSE.06 cells, TSE cells were serially passaged in T-75 flasks in medium containing dialyzed fetal bovine serum and progressively lower GLN concentrations. At each passage, confluent flasks were harvested, and half of the cells were seeded into a flask containing medium with a GLN concentration half that in the preceding culture. After approximately 4 months, cells growing in a GLN concentration of 0.06 mM were obtained and named TSE.06 cells. These cells were maintained in medium containing dialyzed fetal bovine serum and 0.06 mM L-GLN. For GLN deprivation and concentration-response experiments, cells were plated at various dilutions in 10-cm tissue culture dishes with complete DMEM and allowed to plate overnight (TSE, HBL100, T47D, and TSE.06) or for approximately 42 h (SKR3 and BT483) before being rinsed twice with GLN-free medium (identical to complete DMEM, except supplemented with 10% dialyzed fetal bovine serum and no GLN) and fed with GLN-free media containing amino acids or drugs as described in the figure legends. Plating dilutions ranged from 1:3 (HBL100) to 1:6 (TSE) and were chosen to obtain cultures that were between 30 and 40% confluent at the initiation of GLN deprivation. SKBR3 and BT483 were allowed to plate for longer periods because they exhibit relatively slow attachment, spreading, and growth rates.
Cell Proliferation Assays-Subconfluent cells growing in complete DMEM were harvested, counted, and diluted in GLN-free medium. Cells were seeded in 96-well plates at approximately 4 ϫ 10 4 cells/well (HBL100) or 1 ϫ 10 4 cells/well (TSE) and allowed to attach overnight in GLN-free medium. The next day, equal volumes of GLN-free media supplemented with 2ϫ concentrations of GLN were then added to wells so that the final GLN concentrations were between 0 and 4 mM. At the times indicated after feeding, relative cell densities were determined using the colorimetric tetrazolium-salt 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide proliferation assay (26,27). Absorbance values (A) were calculated from OD values measured using a 550-nm wavelength filter with a 650-nm reference filter (A ϭ A 550 nm Ϫ A 650 nm ).
RNA Stability Assays-TSE cells were seeded in 100-mm plates at a 1:6 dilution in complete DMEM and allowed to plate overnight (18 h). These cultures were then rinsed twice with GLN-free medium, fed with GLN-free medium, and incubated for 19 h before being fed with GLNfree media supplemented with 0 or 4 mM GLN and with or without 5 g/ml actinomycin D or with and without 100 M 5,6-dichloro-1-b-Dribofuranosylbenzimidazole (DRB). Actinomycin D was added from a 5 mg/ml stock in absolute ethanol, and ethanol carrier (0.1% final concentration by volume) was added to control cultures. DRB was added from 100 mM stock in Me 2 SO and Me 2 SO carrier (0.1% final concentration by volume) was added to control cultures. At various times after feeding, cultures were harvested and total RNA isolated as described for Northern blotting.
Transcription Assay-Nuclear run-on assays were performed using a modification of published methods (30) employing RNeasy™ total RNA extraction kits (Qiagen) to isolate nuclear RNA after in vitro transcription and ExpressHyb™ hybridization solution (CLONTECH) for capture reactions. For in vitro transcription reactions, 2-3 ϫ 10 7 nuclei were incubated for 30 min at 30°C in transcription buffer (30) containing 250 Ci of [␣-32 P]UTP. Following chromatin disruption with high salt buffer, DNase treatment, and isopropyl alcohol precipitation, RNA was resuspended in 0.1% SDS, diluted with lysis buffer RTL (Qiagen), and then purified using the RNeasy™ total RNA extraction kit, following the manufacturer's instructions. Transcripts were captured by linearized and denatured plasmids (described above for Northern blotting) immobilized on nylon membranes at 2 g/slot. Capture membranes were blocked for 15-30 min in ExpressHyb™ hybridization solution at 65°C and then contacted with 1 ϫ 10 7 cpm/ml of purified in vitro labeled RNA diluted in ExpressHyb™ hybridization solution for 30 h at 65°C. After extensive washing at 65°C with 1ϫ SSC containing 0.1% SDS, membranes were treated with 50 g/ml RNase A (Qiagen) for 30 min at room temperature and then washed at room temperature with 1ϫ SSC containing 0.1% SDS. Captured transcripts were detected by contacting membranes to hypersensitive x-ray film (Reflection™, NEN Life Science Products), and autoradiographic images were quantified using a laser densitometer (Molecular Dynamics).

RESULTS
HBL100 and TSE Cells Are Highly Dependent upon Ambient GLN for Growth and Viability-Previous work from our laboratory examined the effect of medium GLN concentration on the growth and death of a series of human cell lines (25). For all these cell lines, proliferation and ultimate cell density were dependent on ambient GLN concentration. Increased initial GLN concentrations supported faster growth rates and higher ultimate densities. The cell lines TSE and HBL100 demonstrated the greatest dependence upon GLN for growth and viability. Growth and death curves of TSE and HBL100 cells are shown in Fig. 1. Exponentially growing cells were seeded at low density in GLN-free medium, allowed to attach overnight, and then fed with media containing various concentrations of GLN to obtain final GLN concentrations between 0 and 4 mM. Within 1 day after FIG. 1. Glutamine-dependent growth and viability of the human breast cell lines HBL100 and TSE. Cells were seeded in 96-well plates at approximately 4 ϫ 10 4 cells/well (HBL100) or 1 ϫ 10 4 cells/ well (TSE) in GLN-free medium and allowed to attach overnight. Equal volumes of media were then added to wells so that the final GLN concentrations were between 0 and 4 mM. At the times indicated after feeding, relative cell densities were quantified using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay as described under "Experimental Procedures." Concentrations of GLN were as follows: 0 mM (Ⅺ), 0.25 mM (E), 0.5 mM (छ), 1.0 mM (‚), 2.0 mM (ƒ), and 4.0 mM ().
feeding with GLN, cell densities began to increase in a GLN-dependent fashion. For HBL100 and TSE cells, GLN concentrations of 2.0 and 1.0 mM, respectively, were required to support maximal growth. The results shown are in keeping with the results of previous work, which determined that 0.8 and 0.6 mM GLN was required to support half-maximal growth rates of HBL100 and TSE cells, respectively (25). GLN deprivation caused the gradual death of these cells over the course of 5 days, as exhibited by a decline in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide absorbance and cell confluence, as well as the appearance of detached cells and debris.
GADD45 and GADD153 mRNA Levels Are Rapidly and Dramatically Induced during GLN Starvation of HBL100 and TSE Cells-Because TSE and HBL100 cells exhibited pronounced GLN dependence for growth and viability, these two cell lines were chosen to examine the induction of mRNAs corresponding to several stress response genes by acute GLN deprivation. Subconfluent cells were rinsed and fed with GLN-free medium, and the levels of GADD45, GADD153, and GRP78 mRNAs were determined in attached cells after 0 and 45 min and 1.5, 3, 6, 12, and 24 h of GLN deprivation (Fig. 2). GAPDH mRNA levels were also determined and used as a normalization factor to correct for RNA loading and transfer variations between samples. GLN deprivation caused a rapid and dramatic induction of both GADD mRNAs and a consistent, but lesser, induction of GRP78 mRNA. GADD mRNAs were appreciably elevated within 1.5 h of GLN starvation. In the case of HBL100 cells, the GAPDH mRNA-normalized level of GADD45 mRNA reached a maximum of 20-fold over the initial level at 12 h, and the level of GADD153 mRNA reached a maximum of 12-fold above the initial level at 6 h. In contrast, the GRP78 mRNA level peaked at only 1.8-fold greater than the initial level at 6 h. GAPDH mRNA levels were not consistently affected by GLN deprivation.
In TSE cells, GADD mRNAs were also discernibly elevated within 1.5 h of GLN starvation. In contrast to HBL100 cells, the level of all three mRNAs increased linearly with time in TSE cells, apparently not reaching maximum levels. At 24 h, GADD45 and GADD153 mRNA was increased by approximately 160-and 180-fold over the initial levels, respectively. GRP78 mRNA reached only 3.5-fold the initial level at 24 h. In contrast to HBL100 cells, the apparent level of GAPDH mRNA in TSE declined in a roughly linear fashion to approximately 31% of the initial value. Because of this, the increases in normalized GADD and GRP78 mRNA levels may overestimate the true induction of expression. Nonetheless, GLN deprivation caused the levels GADD45 and GADD153 mRNA to increase by at least 50-and 55-fold, respectively. Rinsing HBL100 or TSE cells with GLNfree medium and then feeding with medium containing a normal amount of GLN (4 mM) did not cause an observable induction of GADD mRNAs (see below, and data not shown).
GADD45, GADD153, and GRP78 mRNA Levels Are Inversely Related to Ambient GLN Concentration and Coincide with GLN Dependence of Growth-To determine the dose response of induction of these stress-response mRNAs, subconfluent HBL100 and TSE cells were rinsed with GLN-free medium and fed with media containing serial dilutions of GLN from 4.0 to 0.06 mM. At 24 h after feeding, levels of GADD45, GADD153, and GRP78 mRNAs were determined and normalized to GAPDH mRNA levels. Media containing reduced concentrations GLN elicited a dramatic increase of both GADD mRNA levels and a small induction of GRP78 mRNA (Fig. 3). In the case of HBL100 cells, the GAPDH mRNA-normalized level of GADD45 mRNA was increased to a maximum of approximately 40-fold compared with the level in cells incubated with 4.0 mM GLN, and the level of GADD153 was increased to a maximum of approximately 20-fold. For both GADD mRNAs, maximum levels were observed in cells incubated with 0.5 mM GLN. Lower GLN concentrations actually caused a slight decrease of these mRNA levels from the maximum at 0.5 mM GLN. In contrast, GRP78 mRNA level was increased by a maximum of only 1.2-fold at 0.5 mM GLN. Interpolation of results suggested that half-maximal induction of these mRNAs occurs between 1.0 and 0.5 mM GLN. GAPDH mRNA levels were not consistently affected by GLN concentration.
In TSE cells, GADD45 mRNA induction was greatest, approximately 500-fold the level in 4 mM GLN-fed cells, at the lowest concentration of GLN (0.06 mM). The level of GADD153 mRNA also was greatest, approximately 270-fold, at 0.06 mM GLN. GRP78 mRNA levels were increased by a maximum of only 4-fold at 0.06 mM GLN. Interpolation of the results suggested that half-maximal inductions of these mRNAs occurs between 0.5 and 0.25 mM GLN. In contrast to HBL100 cells, GAPDH mRNA levels were affected by GLN deprivation. The apparent level of GAPDH mRNA in TSE cells was reduced in cultures fed with media containing less than 1.0 mM GLN, declining to approximately 38% of the level in 4 mM cells at 0.06 mM. Again, the increases in normalized GADD and GRP78 mRNA levels may therefore overestimate the true induction of expression, but GLN deprivation did cause the levels of GADD45 and GADD153 mRNA to increase by at least 190-and 100-fold, respectively.
It should be noted that rinsing of TSE or HBL100 cell with GLN-free medium and then feeding with GLN-replete medium did not cause any appreciable accumulation of GADD45, GADD153, or GRP78 mRNAs after 24 h. In addition, time course experiments were performed that examined the kinetics FIG. 2. Induction of GADD45, GADD153, and GRP78 mRNA expression by GLN starvation. Cells were seeded in medium containing 4 mM GLN, allowed to plate overnight, and then rinsed and fed with GLN-free medium. At the indicated times after feeding, total RNA was isolated from cells, equal amounts of total RNA were electrophoresed (10 g/lane), and Northern blotting was performed as described under "Experimental Procedures," utilizing cDNA-derived probes for specific genes as indicated.
of induction for GADD45 and GADD153 mRNAs in TSE cells fed with media containing various GLN concentrations after being rinsed with GLN-free medium, and no transient induction of GADD mRNA levels was observed in cells fed with GLN-replete medium (data not shown).

Induction of GADD45 and GADD153 Levels Correlates with the GLN Dependence Exhibited by Several Human Breast Cell
Lines-In a previous report, we characterized the GLN dependence and the GLN transporter characteristics for a series of human breast carcinoma cell lines (25). A direct correlation was found between two parameters: the GLN ED 50 for growth and the K m for GLN of the System ASC transporter. These represent the concentration of GLN required for half-maximal growth of each cell line and the concentration of GLN at which each cell line's transporter is able to transport GLN at a halfmaximal rate, respectively. The induction of GADD45, GADD153, and GRP78 by GLN deprivation was examined in several of these cell lines, including HBL100 and TSE cells as well as a derivative of TSE cells that was gradually adapted to growth in medium containing a low GLN concentration (0.  Table I. Human breast cell lines that are less GLN-sensitive exhibited inductions of GADD45 and GADD153 mRNA levels by GLN deprivation that were less pronounced and occurred at lower GLN concentrations than the inductions in TSE or HBL100 cells. In addition, TSE cells adapted to grow in medium containing only 0.06 mM GLN (TSE.06 cells) no longer exhibited a striking response to GLN deprivation.

Transcription of GADD45, GADD153, and GRP78 Genes Is Increased by No More than 2-5-Fold by GLN Deprivation-
The large increase in GADD45 and GADD153 expression caused by GLN deprivation could be attributed to increased transcription rates, increased mRNA stability, or a combination of these mechanisms. In order to test the effect of GLN upon gene transcription, subconfluent TSE cells were rinsed and fed with GLN-free medium with or without the addition of 4 mM GLN, and nuclei were isolated 3 h after feeding. Capture membranes were hybridized with equal activities of purified RNA labeled during in vitro, run-on transcription with nuclei from GLNdeprived and GLN-fed cells. A representative experiment is shown in Fig. 4. Compared with GAPDH, the amount of radioactively labeled transcripts captured by immobilized GADD45, GADD153, and GRP78 cDNAs was relatively small. When the RNA was derived from nuclei of GLN-deprived cells, the amount of radioactivity captured by the GAPDH cDNA was slightly reduced, whereas that captured by GADD45, GADD153, and GRP78 cDNAs was increased by 4-, 3-, and 2-fold, respectively. This analysis was repeated three times  with similar results. The estimated increases in GADD45, GADD153, and GRP78 transcription by GLN deprivation varied but were always within 2-5-fold. This analysis suggested that GLN deprivation causes up-regulation of GADD45, GADD153, and GRP78 transcription. However, the marked increase in GADD45 and GADD153 mRNA levels observed cannot be fully attributed to this mechanism. In contrast, the increase of GRP78 mRNA levels may be due to this mechanism alone. GLN Causes Rapid Degradation of GADD45 and GADD153 mRNA-In order to determine whether GLN may affect the stability of GADD45 and GADD153 mRNA, the decline of GADD45 and GADD153 mRNA levels in GLN-starved cells was examined following GLN repletion and/or the addition of two inhibitors of transcription, actinomycin D and DRB. In separate experiments, subconfluent TSE cells were starved for GLN overnight (approximately 18 h) to bring about the accumulation of high GADD45 and GADD153 mRNA levels. In the first experiment, GLN-deprived cells were then fed with GLNfree medium to which 0 or 4 mM GLN was added and 5 g/ml actinomycin D or 0.1% ethanol carrier. At the various times after feeding, relative mRNA levels were calculated by comparison of GAPDH-normalized values with the level observed in cells at time 0 (Fig. 5A). GLN repletion elicited a rapid decline in GADD45 and GADD153 mRNA contents that was not greatly affected by actinomycin D. Half-lives of mRNA species were estimated by least-squares analysis of ln(GAPDH-normalized mRNA levels) versus time, obtaining a decay constant from the slope of the linear portion of each curve. After feeding with GLN, GADD45 mRNA levels decayed with apparent halflives of 45 and 29 min with and without actinomycin D, respectively. Likewise, GADD153 mRNA levels in GLN-fed cells decayed with apparent half-lives of 36 and 26 min with and FIG . 4. Effect of GLN deprivation on GADD45, GADD153, and GRP78 transcription rates. TSE cells were plated in 4 mM GLNcontaining medium overnight and then rinsed and fed with medium containing 0 or 4 mM GLN. Three hours after feeding, cells were harvested, and nuclei were isolated and cryopreserved. Transcription from nuclei (2 ϫ 10 7 ) was continued for 30 min at 30°C in the presence of [ 32 P]UTP. Capture membranes were prepared by binding 2 g each of linearized and denatured plasmids containing the cDNA inserts indicated in an array as follows: GADD45 in the upper left position, with GADD153 and GRP78 below; GAPDH in the upper right position with pBS below that (pBS represents pBluescript SKϩ). Two identical capture membranes (shown side by side) were hybridized for 42 h with equal activities (2 ϫ 10 7 cpm) of purified RNA from cells incubated with (ϩ) and without (Ϫ) GLN, followed by washing, RNase treatment, and autoradiography.
FIG . 5. Effect of GLN on GADD45, GADD153, and GRP78 mRNA decay. Subconfluent TSE cells were starved for GLN for 19 h and then fed with GLN-free medium to which 0 or 4 mM GLN was added, which contained an inhibitor of transcription or carrier alone. A, 5 g/ml actinomycin D (ActD) was added to inhibit transcription, and 0.1% ethanol carrier (EtOH) was added to control cultures. B, 100 M DRB was added to inhibit transcription, and 0.1% Me 2 SO (DMSO) was added to control cultures. At the indicated times after feeding, total RNA was isolated, and quantitative Northern blotting was performed. Relative mRNA levels were calculated by comparison of GAPDH-normalized values with the level observed in cells at time 0. without actinomycin D, respectively.
In contrast, when cells were fed with GLN-free medium, GADD45 mRNA levels were maintained both in the absence and presence of actinomycin D. In fact, except for the 8-h sample, GAPDH-normalized GADD45 mRNA levels seemed to increase slightly after feeding with GLN-free medium containing actinomycin D. This indicates that the decay rate of GADD45 mRNA was similar to that of GAPDH mRNA. In these cells, GAPDH mRNA half-life was estimated to be approximately 13 h. Comparing this figure to the half-life of GADD45 mRNA observed in the presence of GLN (45 min), it can be estimated that the decay of GADD45 mRNA was accelerated approximately 17-fold by ambient GLN. GADD153 mRNA levels were also maintained after feeding cells with GLN-free medium and ethanol carrier. When actinomycin D was included, the apparent half-life of GADD153 mRNA was 2.8 h. This contrasts with a half-life of 36 min observed in the presence of GLN and actinomycin D. Thus, the GADD153 mRNA half-life was accelerated approximately 5-fold by ambient GLN. In contrast, in the presence of actinomycin D, the decay of GRP78 mRNA accelerated by less than 2-fold by ambient GLN. The apparent half-life of GRP78 mRNA was 9.2 h in GLN-free medium and 4.8 h in the presence of GLN. This comparison, together with the expression of GRP78 mRNA observed in the ethanol control cultures, suggests that the effect of GLN upon GRP78 mRNA expression is equally attributable to transcriptional and post-transcriptional mechanisms.
Nearly identical results were obtained when this experimental procedure was repeated using DRB, rather than actinomycin D, to block transcription (Fig. 5B). This experiment confirmed the rapid decay of GADD45 and GADD153 mRNA levels in cells fed with 4 mM GLN (and treated with 01% Me 2 SO carrier). In this case, GADD45 and GADD153 mRNA levels as well as GRP78 levels continued to increase for 8, 4, and 24 h after refeeding the cells with GLN-free medium with Me 2 SO carrier. The reason for this is not apparent, for the cells were starved for GLN for approximately 18 h prior to refeeding. DRB effectively inhibited the further increase in these mRNA levels in continuously GLN-starved cells. After feeding with 4 mM GLN, GADD45 mRNA levels decayed with apparent half-lives of 53 and 26 min with and without DRB, respectively. Likewise, GADD153 mRNA levels in GLN-fed cells decayed with apparent half-lives of 38 and 28 min with and without DRB, respectively. Feeding with GLN and Me 2 SO carrier caused GRP78 mRNA levels to transiently increase and then decrease to approximately 40% of the level at time 0. In the presence of 4 mM GLN and DRB, GRP78 mRNA decayed with an apparent halflife of 3.6 h. When cells were refed with GLN-free medium and DRB, the GADD45 mRNA levels were maintained and slightly increased relative to GAPDH mRNA levels. Again, this indicated that the decay rate of GADD45 mRNA was similar to that of GAPDH mRNA. In these cells, GAPDH mRNA half-life was estimated to be approximately 11 h. Comparing this value to the half-life of GADD45 mRNA in the presence of 4 mM GLN and DRB (53 min), it can be estimated that GLN feeding caused an approximately 12-fold increase in the GADD45 mRNA decay rate. In the presence of DRB, GADD153 mRNA decayed with an apparent half-life of 2.3 h when cells were refed with GLNfree medium. By comparison with the figure obtained in the presence of GLN and DRB (38 min), it is apparent that GLN caused an approximate 4-fold increase in the GADD153 decay rate. Under GLN-free conditions and DRB, GRP78 mRNA decayed with an apparent half-life of 6 h, which was approximately 65% greater than the half-life obtained in the presence of ambient GLN. Thus, all the findings obtained using actinomycin D to block transcription were confirmed by the use of DRB. DISCUSSION The data presented here describe a rapid and pronounced response to GLN starvation; namely, the induction of GADD45 and GADD153 mRNA expression. This response was particularly acute in two human breast cell lines (HBL100 and TSE), which exhibit relatively high rates of GLN utilization and extreme dependence upon GLN for growth and viability (25). HBL100 cells were originally obtained from a mother's milk shortly after giving birth (31). This cell line is considered to be untransformed but can become tumorigenic during extensive in vitro culturing (32). TSE cells were derived from the tumor of a ductal adenocarcinoma patient. TSE cells exhibit an epitheliod morphology and form tumors in athymic nude mice (data not shown). The induction of GADD45 and GADD153 mRNA expression was lesser in breast cell lines that are less GLN-dependent. In general, the half-maximal inductions roughly coincided with GLN dependences for growth and GLN transporter affinities (Table I). This suggests that this response coincides with diminished GLN influx or, conceivably, a lack of GLN transporter occupancy. However, the results with TSE.06 cells suggest that transporter affinity does not determine the GLN concentration at which the response is half-maximal. TSE.06 cells exhibit very little GLN-dependence for growth but retain a GLN-transporter affinity that is not very different from that of the highly GLN-dependent parental TSE cells. The K m of the ASC GLN transporter expressed by TSE.06 cells was determined to be 0.19 mM, whereas that of the parental TSE cells is 0.39 mM. In contrast, adaptation to growth in low-GLN medium caused the GLN ED 50 for growth to be reduced by 10-fold in TSE.06 cells compared with parental TSE cells. Consequently, the half-maximal induction of GADD45 and GADD153 expression in TSE.06 cells was observed at approximately 0.06 mM GLN (Table I). By comparison with TSE cells, the induction of GADD mRNA expression by TSE.06 cells might be expected to be half-maximal at 0.3 or 0.4 mM if the GLN transporter affinity determined this response. In addition, the extent of GADD mRNA inductions are reduced by more than 1 order of magnitude in TSE.06 cells compared with the parent line.
Whether the up-regulation of GADD45 and/or GADD153 plays any causal role in the growth arrest of cell death that results when HBL100 and TSE cells are deprived of GLN has yet to be tested. There are indications that these GADD45 and C/EBPhomologous proteins possess directly antiproliferative or apoptotic functions. Several previous studies have implicated GADD45 expression in the mechanism of growth arrest triggered by p53 expression (2,10,(13)(14)(15). GADD153 expression has also been implicated in growth arrest and apoptosis (4,11,12). Conversely, GADD45 and/or GADD153 could possess functions analogous to traditional stress-response genes, serving to protect cells from stress-induced damage and/or aiding the recovery of normal cellular functions following stress. Indeed, growth arrest could itself serve as a protective mechanism, by conserving energy and halting DNA synthesis in a time of glutamine scarcity.
The effect of GLN deprivation on the expression of another stress-response gene, GRP78, was also examined. In contrast to GADD45 and GADD153 mRNA, GRP78 mRNA levels were increased only marginally in GLN-starved cells. This was surprising, given that many genotoxic agents that induce GADD153 expression have comparable effects on GRP78 expression (23,33,34). In particular, GADD153 and GRP78 are expressed in response to agents that interfere with glycoprotein expression, an observation that led to the conclusion that both genes respond primarily to ER stress (16). Moreover, exogenous expression of GRP78/BiP depressed the induction of GADD153 expression in response to several agents that cause ER stress as well as methyl methanesulfonate, an agent thought to induce GADD153 expression by causing DNA damage (16).
GADD153 expression is induced by deprivation of cells with several amino acids. Marten et al. (35) demonstrated the induction of GADD153 mRNA levels in rat hepatoma cells by deprivation of phenylalanine, methionine, leucine, and tryptophan. In these cells, tryptophan deprivation caused the greatest induction, which was approximately 40-fold. Bruhat et al. (24) demonstrated that leucine starvation caused a 30-fold up-regulation of GADD153 expression in HeLa, HepG2, and Caco-2 cells. These authors also demonstrated that deprivation of several amino acids caused GADD153 promoter activity to be increased by up to 8-fold in reporter-transfected HeLa cells. GLN deprivation caused GADD153 promoter activity to be increased by approximately 3-fold. This is consistent with the run-on transcription results (Fig. 4), which demonstrated that GLN increased transcription of the GADD153 gene in TSE cells by a similar amount. Likewise, GADD45 and GRP78 transcription were increased by 4-and 2-fold, respectively. Thus the marked increase in GADD45 and GADD153 mRNA levels could not be solely attributed to increased gene transcription rates.
An examination of the decay of GADD45 and GADD153 mRNA in TSE cells suggested that GLN caused a rapid destabilization of these two transcripts (Fig. 5). The level of these mRNAs declined rapidly after GLN repletion, regardless of actinomycin D or DRB treatment. However, these mRNAs decayed relatively slowly when cells were fed with GLN-free media containing inhibitors of transcription. The effect of GLN upon mRNA decay rate was most pronounced for GADD45. The decay of this mRNA was accelerated by 12-and 17-fold by GLN. GLN had a lesser, but still pronounced, effect on GADD153 mRNA decay, accelerating its decay by 4-and 5-fold. GLN had a lesser effect on GRP78 mRNA decay, increasing its decay by less than 2-fold. Thus, the increases in GADD45, GADD153, and GRP78 mRNA levels produced by GLN deprivation can be attributed to both increases in gene transcription rates and decreases in mRNA decay rates. In the case of GADD153 and GADD45, mRNA stabilization is the predominant cause of elevated mRNA levels. This is particularly true for GADD45 mRNA, which is dramatically stabilized in GLN-deprived cells.
Thus, the most striking effect of GLN deprivation observed in these cells is the pronounced inhibition of GADD45 mRNA decay. Computer analysis of the 3Ј-UTR of human GADD45 mRNA sequence shows that this message can assume a complex secondary configuration, which includes several stem-loop structures (data not shown). GADD45 mRNA stability may be affected by the activity of an RNA-binding protein, which interacts with one of these structures within the 3Ј-UTR of this mRNA. Several unstable mRNAs contain a sequence element known as an AU-rich element (ARE), which when bound by an ARE binding factor (AUF1) causes rapid degradation of these mRNAs (36). AREs contain multiple copies of the sequence AUUUA, and two groups have determined that the consensus ARE is composed of the sequence UUAUUUAUU (37,38). One copy of this element is only weakly destabilizing, but two or more repeats of this element cause pronounced destabilization. It should be noted that the 3Ј-UTR of GADD45 mRNA is relatively AU rich (68%). GADD45 does contain two AUUUA sequences in its 3Ј-UTR at bases 1060 and 1278. However, neither of these match the nine-base consensus; the upstream element is UAAUUUAGA, and the downstream element is UUAUUUAUG. Thus, GADD45 contains only a single "ARElike" element. We have not found any other known RPB recognition sequence element in the GADD45 cDNA sequence.
The mechanism(s) by which GLN depletion and subsequent cellular stresses induce the transcription of and stabilize mRNAs for the growth arrest genes GADD45, GADD153, and GRP78 mRNAs remains to be determined. It is conceivable that completely separate mechanisms underlie these effects. Nonetheless, the antiproliferative nature of GADD45 and GADD153 suggests that the rapidly and dramatically up-regulated expression of these genes represents a potential molecular basis for the long observed yet poorly understood GLN dependence of mammalian cells in culture. Alternatively, the genes may serve to protect cells from the detrimental effects of GLN deprivation.


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Mutational Analysis of the Hydrolytic Activity of Yeast RNA Polymerase III*

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309–325 gave slightly elevated nuclease activity. In region 367–376, two mutations gave 12–15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056–9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli β-subunit sequences between Rifr clusters I and II, produce changes in the relative yields of A- and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T5 terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3′-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309 -325 gave slightly elevated nuclease activity. In region 367-376, two mutations gave 12-15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056 -9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli ␤-subunit sequences between Rif r clusters I and II, produce changes in the relative yields of A-and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T 5 terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.
The elongation phase of transcription by DNA-dependent RNA polymerases has long been viewed as a repetitive sequence of choices between two alternative outcomes: addition of another nucleotide to the growing RNA or dissociation of enzyme and RNA from the DNA template, resulting in termination (2). More recently, this interplay was seen to involve the additional options of pausing induced by certain template sequences and resection of transcript 3Ј termini, an exonuclease reaction triggered by both spontaneous pausing and artificially arrested elongation (3,4).
A common element in pausing, termination, and 3Ј-exonuclease action is their tendency to occur preferentially when the elongating polymerase has just incorporated several uridylate residues into RNA. Eukaryotic RNA polymerases II and III undergo spontaneous arrest and long pausing, respectively, at U 2 or longer tracts (5,6), whereas the sites for pol III 1 transcription termination and Escherichia coli (Rho-independent) transcription termination both occur following long U tracts (7,8).
In earlier studies of in vivo termination by yeast RNA polymerase III, we found that genetic alteration of the second largest subunit can change this enzyme's ability to continue transcription downstream of a U 5 tract. Mutant polymerases with increased read-through of an intronic U 5 sequence in the SUP4 UIV allele can efficiently produce biologically active suppressor tRNA Tyr (9,10). These mutant polymerases also read through the U 5 tract in SUP4⌬94 that is placed at the location of the normal U 7 GU 6 terminator, producing abnormally long pre-tRNAs that are not matured to functional tRNA Tyr . By in vivo mutant screening with these same two SUP4 alleles, a large number of second-largest subunit mutations having the opposite phenotype were also obtained. These have been designated increased termination mutations (10).
RNA polymerase III preparations isolated from several yeast strains bearing increased or decreased termination mutations have an altered response to several U-rich pause sites within the SUP4 template (11). Because of our previous observation (12) that the amounts of mono-and dinucleotide exonuclease products released concurrently with the transcription reaction are directly proportional to the content of oligo(U) tracts in the transcript, we have characterized the set of ret1 mutations in the second largest pol III subunit for the specific activity and substrate preference of the 3Ј-exonuclease of each mutant pol III. We made analytical measurements of the dinucleotide products released during transcription by the mutant yeast enzymes. As templates, we employed the SUP4-o yeast tRNA-Tyr gene, which contains three internal pause sites and a U 7 GU 6 terminator, as well as a synthetically constructed chimeric tRNA Tyr /tRNA Leu tRNA gene. The latter template contained a U 6 terminator between the A and B block internal control regions as well as the normal U 7 terminator at the end of the tRNA 3 Leu gene. For each mutant RNA polymerase III transcribing these two DNA templates, we measured both the total amount of dinucleotide released per transcript completed and the ratio between released pUpU and released pGpU.
Of the 25 ret1 alleles studied, 11 had near normal rates of dinucleotide release, one had significantly slower cleavage, and the remainder had rates of RNA cleavage ranging from 2 to 60 times that of the wild type. The largest effects were produced by mutations in two conserved motifs: DDRDYVGNKR, between amino acids 367 and 376, and GEMERDCYIA, between amino acid 1063 and 1072 (10). Qualitative effects upon the release of pGpU relative to pUpU were observed for mutations in the region from amino acids 476 to 485 (analogous to Rif r cluster II of E. coli rpoB (13)).

MATERIALS AND METHODS
Reagents and Enzymes-[␥-32 P]ATP, [␣-32 P]UTP, fast protein liquid chromatography-purified ribo-and deoxy-NTPs, and the Sculptor TM in vitro mutagenesis system were purchased from Amersham Pharmacia Biotech. Ribo-ApU and UpA were obtained from Sigma, and other ribo-oligonucleotides were from Oligos Etc. Inc. M-280 streptavidin-Dynabeads were purchased from Dynal, Inc. DNase-and RNase-free bovine serum albumin was purchased from Roche Molecular Biochemi-cals. Vent DNA polymerase, T4 DNA polymerase, and T 4 polynucleotide kinase were obtained from New England Biolabs Inc. The QuikChange TM site-directed mutagenesis Kit was purchased from Stratagene. RNase-free solutions, glassware, and plasticware used in all experiments were prepared as described (14).
DNA Templates for Transcription Reactions-Linear doublestranded DNA fragments ϳ400 -500 base pairs in length containing SUP4 tRNA Tyr were obtained by polymerase chain reaction amplification using the M13mp18 plasmid carrying the 256-base pair BamHI-BamHI SUP4 tRNA Tyr fragment (15) as a template. To obtain a pol III template lacking oligo(A) sequences prior to the terminator, a chimeric gene was constructed by placing an artificial terminator between the synthetic A and B blocks of the yeast tRNA 3 Leu gene with SUP4 tRNA Tyr sequences preceding the A block. The ϳ1300-base pair EcoRI-PstI fragment was subcloned into plasmid pUC119, and the resulting plasmid was used as a template in polymerase chain reaction amplification to produce biotinylated DNA fragments containing a chimeric gene as described (12). These linear double-stranded DNA templates, immobilized on magnetic beads through streptavidin-biotin interactions, were used in all transcription experiments. The average concentration of template was ϳ 300 pmol/ml of magnetic beads.
Cleavage Concurrent with Transcription of the SUP4 tRNA Tyr Gene-Yeast P-11 extract, prepared as described (11), but substituting Sephadex G-10 for Sephadex G-50 chromatography, was used as a source of RNA polymerase III and its transcription factors. Ternary complexes of wild-type RNA polymerase III stalled at position ϩ17 were formed in a 15-l reaction mixture containing 5 l of P-11 extract, 3 pM linear double-stranded SUP4 template attached to the magnetic beads, 0.5 mM ATP, 0.5 mM CTP, and 0.5 mM UTP in transcription buffer (20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 7 mM MgCl 2 , 3 mM dithiothreitol, and 6% glycerol). Since the specific activities of some of the mutants were considerably lower than that of wild-type enzyme, transcription reactions for those mutants were proportionally scaled up to obtain an intensity of the full-length product signal approximately equal to that of the wild type. After incubation at 25°C for 30 min, ternary complexes were magnetically concentrated and washed three times with an excess volume of washing buffer (20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 3 mM dithiothreitol, 0.25 mg/ml bovine serum albumin, and 10% glycerol). Transcription was then restarted by the addition of 0.2 mM CTP, 0.2 mM ATP, 0.2 mM GTP, 15 M [␣-32 P]UTP, and 0.5 mg/ml heparin to prevent reinitiation. The total volume of each post-initiation reaction was 6 l. After transcription was allowed to proceed for 3 h to ensure that the slowest mutants had finished traversing the SUP4 gene, the reactions were stopped by the addition of EDTA to 20 mM and 1:3 (v/v) 98% formamide. After heating at 95°C for 30 min and magnetic separation, samples were separated on 20% gels as described (12). To avoid possible distortions of the front bands and to verify that the original [␣-32 P]UTP reagent contained no overlapping contaminants, we loaded buffer containing all components except the ternary complex in between sample lanes. Dried gels were then subjected to PhosphorImager analysis.
Cleavage Concurrent with Transcription of the Chimeric Gene-The formation and purification of 17-mer ternary complexes on a chimeric template were performed under the conditions described above. Transcription was restarted by the addition of 0.2 mM CTP, 0.2 mM ATP, 0.4 mM GTP, 2 M [␣-32 P]UTP, and 0.5 mg/ml heparin to prevent reinitiation. The total reaction volume was 6 l. After incubation for 30 min at 25°C, the reactions were stopped and treated as described above.
Elongation Profiles-Wild-type 17-mer ternary complexes were formed in a 15-l reaction mixture containing 5 l of P-11 extract, 3 pM linear double-stranded SUP4 template attached to the magnetic beads, 0.5 mM ATP, 0.5 mM CTP, and 0.3 M [␣-32 P]UTP in transcription buffer (20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 7 mM MgCl 2 , 3 mM dithiothreitol, and 6% glycerol). For mutant polymerases with lower specific activities, transcription reactions were proportionally scaled up. After incubation at 25°C for 30 min, ternary complexes were purified as described above. After resuspension in 60 l of washing buffer, magnetic beads with the attached ternary complexes were distributed to reaction tubes at 10 l/aliquot. Transcription was then restarted by the addition of 0.1 mM CTP, 0.1 mM ATP, 0.2 mM GTP, 0.2 mM UTP, and 0.5 mg/ml heparin to prevent reinitiation. The total volume of each postinitiation reaction was 40 l. After transcription was allowed to proceed for 0, 1, 2, 3, 4, or 5 s, the reactions were stopped by the addition of 40 l of stop mixture (1 mg/ml proteinase K, 1% SDS, 20 mM EDTA, and 0.25 mg/ml carrier RNA) and incubated at 65°C for 30 min. After magnetic separation and ethanol precipitation, samples were resuspended in loading buffer (100 mM sodium acetate (pH 5.5), 8 M urea, and 0.025% xylene cyanol dye) and separated by 10% polyacrylamide gel electrophoresis.
Site-directed Mutagenesis-Mutant R376A was produced according to the procedure of the Amersham Sculptor TM kit. Mutations D370E and D370A were created by the polymerase chain reaction mutagenesis procedure described in the QuikChange TM site-directed mutagenesis kit. Phagemid pSA-70 containing the BamHI-PstI fragment of RET1 (10) was used as a template in all mutagenesis reactions. The 440-base pair MluI-PstI fragment was then used to replace the corresponding wild-type fragment in expression vector pSA-71. The mutations were rechecked by sequencing the final plasmids. In vivo termination phenotypes of the resulting mutant polymerases were checked as described (10).
Oligonucleotide Standards-Labeled markers were obtained as described (16) by phosphorylation of appropriate 5Ј-nonphosphorylated ribo-oligonucleotides with T 4 polynucleotide kinase.

RESULTS
Cleavage during Elongation of the SUP4 tRNA Tyr Gene-To provide a quantitative measurement of 3Ј-exonuclease action concurrent with transcription, we carried out single-round transcription with yeast RNA polymerase III from each of the ret1 mutant alleles. For this purpose, the DNA template was immobilized on magnetic beads, and the magnetically purified transcription complexes were washed free of unbound proteins and nucleic acids as described previously (12). Transcription was started in the presence of ATP, CTP, and UTP, and then the resulting 17-mer complexes were purified by extensive washing of Dynabeads with the attached ternary complexes. Elongation was restarted in the presence of all four NTPs with [␣-32 P]UTP as the radioactive label. After elongation was completed, reactions were stopped and denatured in the presence of formamide. The samples were then loaded onto 20% polyacrylamide gels to detect both the RNA and oligonucleotides made during the transcription reaction.
Cleavage by mutant polymerases during elongation on the SUP4 tRNA Tyr gene is shown in Fig. 1. Under the conditions used, the intensities of cleavage products were ϳ100 times higher than those of full-length RNA. For autoradiographic display of both long and short products separated on the same gel, the bottom part of gel was exposed against the film for only 2 h in comparison with 12 h for the upper part that contains completed RNA molecules.
As shown Fig. 1, many of the mutants investigated have marked differences in their hydrolytic properties compared with that of wild-type RNA polymerase. For example, mutants K310T, V309E, E325K, M312T, and T311K produced considerably more of the pyrimidine dinucleotide (pPypPy) product than did the wild type (Fig. 1, A and B). This increase was even more obvious in the case of mutants D370E and R376A (Fig.  1D) and mutants I1071S and A1075V (Fig. 1E).
For quantitation, the gels were analyzed by a PhosphorImager, permitting a comparison of the amounts of full-length transcript and short cleavage products produced per round of transcription. For every mutant, at least three replicate experiments were done. The results are summarized in Table I. The three parameters we chose typify different aspects of the cleavage reaction. The first of these (Table I, second column), frequency of nucleolytic events per round of transcription, is defined as the ratio between the amounts of pPypPy product and full-length RNA for each mutant. The other parameters were the ratios between pGpU or pApU and the major cleavage product pPypPy. The values in the second and sixth columns were normalized to those for wild-type pol III, set at 100%, whereas the values for dinucleotide ratios were normalized to a wild-type value of 1.0.
pol III enzymes with ret1 mutations between amino acids 300 and 325 performed cleavage several times more frequently than does the wild-type enzyme (Table I). For these, the rela-tive amounts of different dinucleotide products remained about the same as for the wild-type polymerase. The only exception is mutant M312I, which showed a 2-fold increase in the relative production of pApU product.
Mutations in the conserved regions 370 -376 and 1061-1075 caused drastic qualitative and/or quantitative changes in the cleavage reaction. Mutant polymerases from these regions performed cleavage 12-60 times more frequently than did the wild-type enzyme. Amino acid substitutions at the invariant residue Asp-370 led to a reduction in the relative yield of G-containing dinucleotide. Substitution of alanine for aspartate also caused a decrease in the relative production of pApU. Mutant I1071S cleaved 60 times more frequently than did the wild type and produced twice as high an amount of pApU, with no effect on pGpU production. In contrast, mutant A1075V demonstrated a considerably lower relative yield of pGpU product, but did not alter pApU production. Mutant R1061S demonstrated behavior that was similar to that of mutants in region 455-512. While cutting with the same frequency as the wild-type enzyme, it showed a 2-fold increase in the relative production of pApU.
Mutations in region 455-512 exhibited a very specific effect on hydrolytic activity. Mutant polymerases V483D, G485N, and K512N and the double mutant T455I/E478K produced less G-containing products than did the wild-type enzyme. In addition, mutant G485N and double mutant T455I/E478K showed a reduced production of pApU product. The frequency of cleavage events per round of transcription was unchanged for all Reactions were allowed to proceed at 25°C for 3 h. Samples were separated on 20% gels. Film was exposed to the top part of the gel for 12 h and to the bottom part of the gel for 1 h. wt, wild type. these mutants. It is interesting that altered termination mutations in the adjoining region 512-517 showed no changes whatsoever in hydrolytic properties.
Cleavage during Elongation on the Chimeric tRNA Tyr / tRNA Leu tDNA Template-To further simplify the analysis of short cleavage products of varying composition, we analyzed 3Ј-exonuclease products and transcripts made on the chimeric tRNA Tyr /tRNA Leu pol III template (12) that lacks natural arrest sites. In transcribing this template, wild-type ternary complexes elongate synchronously up to the terminator without pausing. Since the artificially constructed terminator (T 7 ) of the chimeric template is not as efficient as the double terminator (T 7 GT 6 ) of the SUP4 tRNA Tyr gene, to prevent substantial "leakage" through the terminator, we performed elongation at a reduced UTP concentration (2 M). Products formed during a single round of elongation on this chimeric template are shown in Fig. 2. Mutants in the region between amino acids 300 and 325 exhibited the expected severalfold increase in cleavage frequency per round of transcription. Interestingly, mutants from region 455-512, which produced less of the Gcontaining cleavage products during transcription of the SUP4 tRNA Tyr gene, showed considerably higher amounts of pGpU product formed on the chimeric template than did the wild-type RNA polymerase. The sizes of the full-length products made by these mutants were also quite different from those made by the wild type. Mutants G485N, K512N, and V483D and double mutant T455I/E478K displayed pausing at sites through which wild-type polymerase and all other mutants elongated smoothly. To determine the positions on the template at which these mutants pause, we performed electrophoretic separation of the products on a long 10% polyacrylamide gel (Fig. 3A). The results show that these mutant polymerases are arrested at or near positions where the RNA transcript has a G residue at its 3Ј terminus. To check if there are any other positions on chimeric template through which these "G mutants" elongate differently from the wild-type enzyme, we carried out elongation kinetics with time intervals as short as 1 s. The results ( Fig. 3B) demonstrate that this pausing at G-associated positions happens throughout the gene, not only near the terminator. The pausing occurs preferentially at G residues that precede a pyrimidine. DISCUSSION The termination of transcription by RNA polymerase III (8,17,18) and the 3Ј-exonuclease activity of this enzyme are both processes that respond strongly to the presence of uridylates at the 3Ј terminus of nascent transcripts. Because of the shared dependence of these two processes upon U residues, we have examined a collection of altered termination mutants in yeast pol III for possible qualitative or quantitative changes in their pol III exonuclease activity. Many of these mutant enzymes have either an enhanced or reduced tendency to pause at short clusters of T residues in the non-template strand (11). Different mutant alleles vary in the time they require to complete transcription of a tDNA template; the number and length of pauses during transcription are by far the most important determinants of the time required for transcription of a tRNA gene (11).
Mutations Quantitatively Affecting Nuclease Activity-There is a distinctive range of mutant nuclease phenotypes within each of the four gene regions from which we have chosen alleles for study (Fig. 4). In the Arg-Lys-rich region 309 -325, all the mutant polymerases except one have elevated nuclease activity; generally, the mutant cleavage frequency is higher than wild type by a factor of 2-3. The relative composition of cleavage products remains the same as for wild-type pol III. Continuing studies on mutants in this region show that they have large effects upon the kinetics of transcript release independently of any change in pol III exonuclease activity.
Within the conserved domain 367 DDRDYVGNKR 376 (Fig. 4), changes in invariant residues Asp-370 and Arg-376 in two cases act to strongly influence the cleavage process, yet these conserved residues are not essential for cleavage itself. Mutagenesis at these positions produced a Ͼ10-fold increase in the ; R, strong reduced termination phenotype; I, strong increased termination phenotype; r, moderately reduced termination phenotype; i, moderately increased termination phenotype; frequency, ratio of pPypPy to full-length product; relative production of pGpU; ratio of pGpU to pPypPy; relative production of pApU, ratio of pApU to pPypPy. number of cleavage events as well as a considerable decrease in the proportion of pGpU and pApU cleavage products. The presence of enhanced nuclease activity following such drastic changes in amino acid sequence (Arg or Asp for Ala) argues against participation of this region in the nuclease active site (1), yet it may be located very near to it.
Similar but even stronger effects were shown by two mutants from the conserved C-terminal region 1061-1081 (Fig. 4). The drastic changes exhibited by mutants I1071S and A1075V both in the frequency of cleavage events and in the production of pGpU and pApU can be explained by specific interactions of these residues with sequences at or near the RNA 3Ј terminus.  In addition, previous studies (11,19) on elongation by RNA polymerase III containing the mutant subunit R1061K/E1081D showed drastically slowed transcription due to pausing of very long duration at the normal (U-rich) pause sites. All of these effects seem entirely consistent with a role of this region, corresponding both in sequence and in placement to region I of the E. coli RpoB protein, in processive elongation.
Mutations Qualitatively Affecting Nuclease Activity-We examined 11 different mutant pol III preparations with substitution mutations between amino acids 455 and 517 (Fig. 4), a region that corresponds in position and sequence to Rif r clusters I and II in the E. coli rpoB gene (13). Approximately half of these mutant polymerases, including all with changes in the region from amino acids 476 to 485, gave altered ratios of pGpU and/or pApU to pUpU hydrolytic release. This effect was particularly strong for mutants T455I/E478K and G485N.
Most of the mutant RNA polymerases with alterations in region 476 -485 showed RNA sequence specificity in their dinucleotide release preference. For the chimeric tRNA Tyr /tRNA Leu tRNA transcript, there was heightened pGpU production by these mutants, whereas with the SUP4 transcript, there was diminished release of pGpU for many of the mutant pol III enzymes with amino acid changes between residues 476 and 512. Between these two DNA templates, there are substantial differences in sequence context for all but one of the GU dinucleotides (Table II). For SUP4, the relative release of pApU was substantially reduced in mutant polymerases T455I/ E478K and G485N, but not in V483D or K512N.
Both the differential responses of mutant polymerases with alterations in region 476 -512 to the SUP4 and tRNA Tyr / tRNA Leu templates and their sequence preferences for exonucleolytic dinucleotide production indicate the existence of baseand amino acid-specific interactions between the conserved protein domain D and sequences at the 3Ј-end of nascent RNA. Earlier elongation studies with the V483D and K512N mutant RNA polymerases and the T455I/E478K double mutant showed that these enzymes had a tendency to pause at novel sites (11,19). When tested on the chimeric tRNA Tyr /tRNA Leu tDNA tem-  plate, these three mutant RNA polymerases and the G485N polymerase all underwent a prolonged arrest at positions corresponding to an incorporated G residue (Fig. 3A). The further observation (Fig. 3B) that transient arrest of these mutant polymerases happens mainly at positions where G is followed by a pyrimidine may be explained by an inability of the RNAbinding region in these polymerases to accommodate consecutive RNA bases that differ in size. Finally, all four of the mutant polymerases studied in Fig. 3 (A and B) exhibit a reduced frequency of termination as compared with the wild type. This may well be an independent consequence of the RNA-binding region alteration that directly affects the termination process (19).
Null Mutations for pol III Exonuclease-A third possible phenotype, one we have not observed for any of the ret1 mutations, would be the total lack of nuclease activity. Recent experiments carried out by Chédin et al. (22) suggest that such a mutation in ret1 would likely be lethal. These authors characterized an altered form of yeast RNA polymerase III, devoid of subunit C11, that does not pause for extended periods at the U-rich pause sites, that exhibits no pol III exonuclease activity in a standard retraction assay (12,20), and that is partially impaired in transcription termination. From their results, they conclude that the C11 subunit allows pol III to switch between an elongation mode and an RNA cleavage mode. Disruption of the gene for pol III subunit C11 is lethal for yeast (22), suggesting that pol III exonuclease is an essential function.
pol III Subunit Function in the 3Ј-Exonuclease Reaction-Because none of the mutant pol III enzymes we studied are devoid of nuclease activity and many have enhanced activity, it seems unlikely that any of the ret1 domains we studied encode the nuclease active site. However, the region encompassing amino acids 455-512 modulates the specificity of nuclease cutting, and the wild-type sequences in regions 367-376 and 1063-1072 appear to down-regulate the catalytic efficiency of pol III nuclease. Subunit C11, on the other hand, appears to activate an intrinsic nuclease activity that is embodied in the pol III core subunits. A number of very interesting questions regarding the relative roles in pol III nuclease of domains within ret1 and subunit C11 can now be approached by genetic means. We hope to find out, for example, whether the hypernuclease phenotypes of any of the mutants at positions 370, 376, 1071, and 1075 are phenotypically epistatic to the absence of subunit C11. Similarly, we might ask whether these mutations suppress the lethality of a C11 gene disruption or the slow growth of a Schizosaccharomyces pombe C11 replacement.
Relationships among Altered Termination, Pausing, and Nuclease Degradation Phenotypes-In relation to the concept of kinetic coupling between transcription elongation and transcription termination (2, 21), many ret1 mutations fall into one of two clear categories. There are a number of mutations, most corresponding in intragenic location to E. coli Rif r clusters I and II, that elongate slowly because of pausing at U-rich sites and that have increased termination and increased overall pol III exonuclease activity. Among these are the mutations E478K, V511E/L516S, N513Y, and R1061K/E1081D (11,19). These behave as expected according to the kinetic coupling model. They spend a longer time elongating across oligo(U) clusters, repeatedly transcribing and degrading RNA in those regions, thus increasing their cross-section for termination.
Quite different behavior was observed for a group of four ret1 mutations centered on residues 483 and 485. These include T455I/E478K, V483D, G485N, and K512N (Table I). The RNA polymerase III from these mutant strains combines a reduced termination phenotype with slow elongation, pauses at G residues, and has a differential lowering of cleavage at G in one context (SUP4) and an increase of cleavage at G in the chimeric template. We attribute the effects of G upon pausing and cleavage in these mutants to the bulkiness of this purine base. The 10-amino acid region encompassing these mutations is conserved within each class of RNA polymerase and contains either 3 or 4 basic amino acid residues in all eukaryotic RNA polymerases and in E. coli RNA polymerase (Fig. 5). We propose that this region engages the nascent RNA-DNA template hybrid very close to its 3Ј (RNA) terminus. These contacts play an important role both in transcription elongation and in the reverse exonucleolytic process. Mutational changes such as V483D and G485N may decrease the size or flexibility of this RNA-binding domain of the protein, resulting in G-dependent pausing and exonuclease changes. At the same time, the changed geometry of this region may cause the enzyme to grasp the RNA-DNA hybrid more firmly, decreasing its tendency to terminate at U 5 sequences.