Identified by Linker Scanning Mutagenesis

Transcription

1 MOLECULAR AND CELLULAR BIOGY, Jan. 1986, p Vol. 6, No /86/ $02.00/0 Copyright 1986, American Society for Microbiology Two Distinct Promoter Elements in the Hman rrna Gene Identified by nker Scanning Mtagenesis MICHELE M. HALTINER, STEPHEN T. SMALE, AND ROBERT TJIAN* Department of Biochemistry, University of California, Berkeley, California Received 15 Jly 1985/Accepted 4 October 1985 A cell-free RNA polymerase I transcription system was sed to evalate the transcription efficiency of 21 linker scanning mtations that span the hman rrna gene promoter. Or analysis revealed the presence of two major control elements, designated the core and pstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (p to -fold) by linker scanning mtations in this region. nker scanning and deletion mtations in the pstream element, located between ncleotides -156 and -107, case a three- to fivefold redction in transcription. Under certain reaction conditions, sch as the presence of a high ratio of protein to template or spplementation of the reaction with partially prified protein fractions, seqences pstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mtant templates is initiated at the correct in vivo start site. To examine the fnctional relationship between the core and the pstream region, mtant promoters were constrcted that alter the orientation, distance, or mltiplicity of these control elements relative to each other. The pstream control element appears to fnction in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, mltiple core elements in close proximity to each other have an inhibitory effect on transcription. The celllar machinery responsible for RNA synthesis is designed to respond to a variety of specific signals so that genes can be transcribed and expressed in a controlled fashion. For example, the synthesis of rrna is sensitive to a variety of physiological signals sch as response to ntrient starvation, reglation by cell cycle, the state of cell proliferation, and invasion by viral infection (6, 9, 17, 24, 26, 27, 31). In addition, an interesting and nexpected property of rrna transcription is the inability of the transcriptional machinery of one species to initiate transcription from the promoter of divergent species. Ths, whereas both sets of rrna genes can be transcribed in an interspecific rodent cell hybrid, the genes from only one species are expressed in rodent-hman cell hybrids (22, 32). This species specificity is mimicked in vitro in that extracts from one species are capable of transcribing only those rdna templates that are derived from the same or closely related species (5, 12, 14). The limited homology in the DNA seqence srronding the transcription start site of mammalian rrna genes sggests that species-specific promoter recognition is likely to involve distinctive rrna promoter elements. Stdies with fractionated enzyme components derived from mammalian cell extracts have allowed the identification of at least two distinct activities that are necesary to reconstitte accrate and efficient initiation of transcription from the hman ribosomal promoter (12, 20, 23). Ths, in addition to the endogenos RNA polymerase I enzyme, promoterselective initiation of transcription reqires the presence of a specific factor, S, that has recently been prified greater than,000-fold and shown to be the factor that confers species-specific promoter recognition to RNA polymerase I (12). As a complementary approach to characterization of transcription factors and in vitro reconstittion experiments, we initiated a systematic analysis of the promoter seqences * Corresponding athor. 227 reqired for RNA polymerase I transcription. We previosly mapped the bondaries of the hman rrna promoter by deletion analysis in a cell-free transcription system (13). These in vitro stdies were sbseqently confirmed in vivo by transient expression in transfected primate cells (29). These stdies show that seqences between -52 and +7 are necessary and sfficient to direct transcription and that flanking seqences from -234 to +18 modlate the efficiency of transcription. In this report, we constrcted a series of clstered point mtations to define and characterize in detail the ciscontrolling elements that specify hman rrna promoter recognition. In addition, we also analyzed the fnctional relationship of an pstream control element to a core element by constrcting promoter mtations that reslt in the dplication of either control element or change the orientation and distance of the pstream element relative to the core element. MATERIALS AND METHODS Recombinant DNA. Restriction enzymes were prchased from Bethesda Research Laboratories, Inc. (Gaithersbrg, Md.) or New England BioLabs (Beverly, Mass.) and sed nder conditions recommended by spplier. Klenow fragment and T4 ligase were prchased from Bethesda Research Laboratories, and T4 kinase was prchased from New England BioLabs. The synthetic oligoncleotide primer was a gift from J. Merryweather and Chiron Corp. Prified SL1 was the generos gift of R. M. Learned. nker scanning mtant (LS) plasmids were constrcted either as described previosly (7) or by a trimoleclar ligation between the small SacI-EcoRI fragment of A3 (3' deletion mtant), the small SacI-SalI fragment of A5 (5' deletion mtant), and the large EcoRI-SalI fragment of pbr322 (7). Doble linker scanning mtant plasmids were constrcted by recombining two linker scanning mtant plasmids at the HincIl site at -170 relative

2 228 HALTINER ET AL. to the RNA start site. 1-UCE was constrcted by inverting the Sacl fragment in DLS -186/-191/-86/-89. All plasmids were seqenced by a direct chemical method (16). Preparation of extract and in vitro transcription. HeLa cells in sspension were grown at 37 C to a density of 4 x 105 to 6 x 105 cells per ml and harvested, and whole-cell extract was prepared by the method of Manley et al. (15). DNA template for in vitro transcription was prepared by digestion of CsCl-prified plasmid DNA with restriction endonclease, followed by extraction with phenol, chloroform-isoamyl alcohol, and ether. The DNA was precipitated with ethanol, sspended in 10 mm Tris hydrochloride (ph 7.5)-l mm EDTA, and qantitated by A260 and by agarose gel electrophoresis. Unless otherwise stated, each transcription reaction mixtre (40,l) contained the following: 8,l of extract, 200 ng of mtant template, 200 ng of wild-type template, 500,M CTP, TTP, and ATP, 50 p.m GTP, 1,Ci of [co-32pjgtp (Amersham Corp., Arlington Heights, Ill.), 5 mm creatine phosphate, 80,g of cx-amanitin per ml, 25 mm Tris chloride (ph 7.9), 6 mm MgCl2, 5 mm dithiothreitol, 50 mm KCl, and 10% glycerol. Transcription was performed at 37 C for 30 min. The reactions were terminated, and the RNA was isolated, glyoxalated, and analyzed by agarose gel electrophoresis and atoradiography. The intensity of the bands was qantitated by densitometry. Transcription reactions to be analyzed by primer extension were the same except that they contained spercoiled template, 500 p.m GTP, and no radioactive isotope. Primer extension reactions were performed as described previosly (29), and the samples were analyzed by denatring acrylamide gel electrophoresis and atoradiography. RESULTS Effect of linker scanning mtations on RNA polymerase I transcription. DNA seqences between -158 and +18 relative to the RNA start site were shown previosly to be sfficient to direct RNA polymerase I transcription at wildtype levels in vitro (13). To map the promoter in greater detail, we constrcted a series of linker scanning mtations which spanned this region. The seqence of each linker scanning mtant promoter was determined (Fig. 1). As indicated, many of the mtants contain small deletions or insertions which mst be taken into consideration in assessing the transcription phenotype. In vitro transcription directed by each mtant template was evalated relative to a wild-type control template in a rnoff assay. Typical rnoff experiments and a smmary of reslts from 20 sch experiments are shown in Fig. 2. Differences in the sizes of the rnoff prodcts are de to variations in the method of constrcting the mtant templates (see above). The analysis of linker scanning mtations allowed s to define the following control sqences: (i) an pstream control element which extends from -156 to -107 and affects transcription by a factor of three- to fivefold; (ii) a core control element which extends from -45 to +18 and affects transcription 2- to -fold. In contrast, seqences pstream of -156, between -107 and -45, and downstream of +18 do not appear to be important for transcription in vitro becase templates bearing mtations in these seqences were transcribed at wild-type levels. It is interesting to note that, in two cases, linker scanning mtations that altered the same stretch of DNA had qite different effects on transcription. For example, LS-32/-24 contains a 1-base-pair insertion while LS - 33/-24 is a perfect sbstittion in the same region of the promoter, yet transcription from LS-32/-24 is at least 20-fold less efficient than that from LS-33/-24. A MOL. CELL. BIOL. similar sitation exists for LS-23/-14 and LS-24/-15, which also differ 20-fold in their effects on transcription. This finding sggests that transcription reqires precise seqence and spacing constraints within the core element. To assess the accracy of transcription initiation from the mtant templates, the 5' termini of the transcripts were mapped by primer extension analysis. Using a synthetic primer that hybridizes to seqences between +50 and +73 on the hman rrna promoter, we observed the expected 73-ncleotide cdna prodct of RNA transcripts from each of the mtant templates (Fig. 3). Occasionally, we observed a weak secondary start that mapped to position -1. Each transcription reaction inclded a psedo-wild-type internal control that contained a 10-ncleotide deletion in the transcribed region and prodced a 63-ncleotide cdna prodct at wild-type levels. These reslts are qantitatively similar to those observed with the rnoff assay. The mtant LS+10/ +20 contains a 1-ncleotide deletion in the transcribed region and directs the synthesis of a correspondingly shortened RNA prodct. Distance reqirements for fnctionality of pstream control element. The seqences between -107 and -45 can be systematically altered inclding some small deletions and insertions withot affecting either the efficiency or the accracy of transcription. We were therefore in a good position to address whether larger variations in the spacing between the pstream and core elements affect the efficiency of transcription. To this end, we constrcted linker scanning mtations that either deleted or dplicated seqences in this interstitial region. The position of the pstream control element relative to the core element in these mtant templates is shown in Fig. 4B. Each of the mtant templates was evalated by both the rnoff assay (Fig. 4a) and the primer extension assay (see Fig. 6). The transcription efficiencies of mtant templates containing internal deletions which position the pstream element closer to the core were compared with both the wild-type template and the corresponding 5' deletion mtants. The reslts indicate that the pstream element is fnctional (i.e., transcription is % of wild-type level) if moved 4 ncleotides closer to the core (LS-86/-73 and LS-107/-94) bt ceases to fnction (i.e., transcription is approximately eqivalent to a deletion mtant lacking the pstream element) if moved.16 ncleotides closer to the core element (LS-98/-73, LS-98/-57, LS-86/-45, LS-98/-45, LS-107/-45, LS-107/-34). Transcription was compared between those mtants which position the pstream element frther from the core element and those linker scanning mtants which inactivate the pstream element. The pstream element appeared fnctional when moved 28 ncleotides frther from the core element, bt not when moved an additional 21 ncleotides. These mtants all promoted faithfl initiation as determined by primer extension analysis (see Fig. 6). The reslts from the analysis of distance alteration mtants are smmarized in Fig. 4B and indicate that large changes (greater than 4 to 28 ncleotides) in the length of DNA separating the pstream element from the core are not tolerated. In contrast, small distance alterations have no apparent effect on the overall transcription efficiency. Effect of mltiple or inverted control elements. To stdy the fnctional relationship between the core and pstream elements, mtations were made which reslted either in the dplication of an element or an inversion of one element relative to the other. Templates containing these mtations were assayed by rnoff transcription and primer extension

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4 LS 230 HALTINER ET AL. A I' l K K' l l i~ jl IN, C - we S->_w-w LSCw.naim- -- n p~ --- ae.u P mm moeqt.-,.. es w ~~~~~~~~~~~~~~~ow - e rw2 r v ~~~~*- 955 MOL. CELL. BIOL B ~v/7/7x7/v777777x I. I. I. I. I. I " I. 'L. 1, I.I1. i. -70! i s -, L I * *, JRBflVSCR/ PJ/ PS9YY c- ndl / I e-7 L31-tIDUbDI-nL i 1I 00 LS-157/-156 I [S-149/ [ LS-137/ LS-130/ LS-I08/ LS-107/-94 n_3 LS-98/-89 LS-86/ LS-74/ L S 6 8/ISJ S57 0 n ( LS-59/ r LS-54/-45 LS-43/ la LS-33/-24 n 20 LS-32/ <1 1S-24/ LS-23/- 14 n9--- <1 LS-9/+1 - < I LS i0/ LS-24/-45 -U FIG. 2. Effect of linker scanning mtations on rrna transcription. (A) Rnoff analysis of RNA transcribed from a wild-type rrna gene (WT) and 21 linker scanning mtant templates (LS). Shown is an atoradiogram of RNA prodcts after gel electrophoresis in 1.2% agarose. The templates were prepared from LS-130/-120, LS-107/-94, LS-59/-49, LS-32/-24, and LS-24/-15 by digestion with PvII and from other LS mtants and a wild-type gene by digestion with PstI. Eqimolar amonts of these templates were sed in the reactions. Template from the wild-type control gene was prepared by digestion of prh5n/s (7) with BamHI and was inclded (in approximately twice the molar

5 VOL. 6, 1986 PROMOTER ELEMENTS IN HUMAN rrna GENE 231 VP, LS - '..am1 - - a. go s-73nt " WT-O ~~~~4 b NN _- * 63NT FIG ll. A..M -.-A.1 A... ::7.-.'. I 4 09 Primer extension analysis of RNA transcripts derived from linker scanning mtant templates. The expected positions of the cdna prodcts are shown for the LS and the IWT (LS+24/+43) on the left, and their sizes are shown on the nrght. Maxam and Gilbert seqencing markers (AG, G) were derived from prh5n/s labeled at the BstEII site. LS+ 10/+20 contains a 1-ncleotide deletion in the transcribed region and gives rise to a shortened cdna prodct. (Fig. 5 and 6). A mtant template that contained two pstream elements in tandem was not transcribed more efficiently than a wild-type template (Fig. 5A). This reslt cold be de to the fact that the second element is 111 ncleotides away from the core and ths may be ot of range to fnction or that only the most proximal element can be fnctional. A doble linker scanning mtant that contained linkers in two mtation-insensitive regions on either side of the pstream element was transcribed somewhat more efficiently (120%) than the wild-type gene bt became significantly weaker (30%) when the segment between the linkers were inverted. This phenotype sggests that the pstream element cannot fnction in an inverted orientation. Again, none of these mtations change the transcription initiation site. Finally, mtations containing two tandem core elements, and ths two corresponding RNA start sites, led to the prodction of two correctly initiated transcripts at sbstantially redced levels as shown by rnoff assay (Fig. 5B) and primer extension analysis (data not shown). For example, the proximal start site in LS+ 10/-45 was transcribed at 10% of wild-type level, bt LS+10/+20 (Fig. 2) and A3 +7 (13) were transcribed at 50% of wild-type level. Similarly, LS + 24/-45 was transcribed at 30% of wild-type level, while LS+24/+43 and A3 +22 were transcribed as efficiently as wild-type templates (Fig. 2) (13). In addition, transcription from the downstream element was redced to a level below that observed with a template containing a 5' deletion to -45. Ths, it appears that transcription initiating within each of these core elements is inhibited by the presence of the other element. This series of mtants sggests that the pstream element is orientation dependent and that the presence of mltiple core elements diminishes the level of transcription. Variable effect of pstream seqence. Althogh the pstream seqences stimlated transcription three- to fivefold in the assays described, we fond that this effect cold vary depending on the particlar extract and on the transcription conditions (Fig. 7). In the crde extract, the effect of the pstream seqences was sensitive to the ratio of template to extract. An example of at least 30 assays is shown in Fig. 7. When a relatively low amont of extract was present in the reaction (lanes 1 throgh 3), a wild-type gene was transcribed only slightly more efficiently than a deletion mtation lacking pstream seqences (A5-83) (7). By contrast, as the ratio of extract to template was increased (lanes 4 throgh 9), the importance of the pstream seqences became more evident and cold reslt in a 10- to 20-fold effect. More importantly, we fond that the pstream seqence cold stimlate transcription to a significantly greater extent (-50-fold) when the extract was spplemented with prified SL1, the species-specific transcription factor (12), and polymerase-containing fractions from a heparin-agarose colmn elted with a 0.2 to 0.5 M NaCl salt step (lane 10). These reslts sggest that the pstream seqences can play a very important role, especially nder conditions when the ratio of protein to template is high. DISCUSSION In this stdy we evalated the in vitro transcription efficiency of varios hman rdna templates containing clstered point mtations that span the promoter region. Or analysis revealed two transcription signals that we desigamont of the Pst templates in this experiment) as an internal control for transcription. The expected positions of rnoff transcripts from the above are depicted by arrows on the left of the gel and the sizes of markers (M) (in ncleotides) on the right. (B) Diagram of the linker scanning mtations showing the position of the Sacl linker in the promoter (O) and their relative efficiencies in the transcription assay. The ratio of each mtant to the wild-type internal control was normalized to the ratio of a wild-type template to the internal control (%). The hatched box represents the pstream control element, and the shaded box represents the core element as defined by linker scanning mtagenesis. The scale refers to the promoter, and the nmbers indicate position relative to the RNA start site which is shown by the heavy black arrow. Note that the exact seqence of the mtant templates is shown in Fig. 1.

6 232 HALTINER ET AL. MOL. CELL. BIOL.,/ ''b e b@/@ -~~~~~,4!,.$/" LSC WT-'-. * o-o -miposow one m * g 4w, - m to h A m., B J....L.A A JfBIA5Ct/qPfl9OiV WILD-TYPE 1S-86/- 73 LS-98-/73 LS-98/-57 1S-86/-45 LS-98/-45 IS- 1 07/-45 LS-107/-34 1S-107/-94 S-54/-94 LS-130/-120 LS-54/-73 LS-137/ _~~W 3 3_~~A 2_~~~~~~~~& - = 4t_-~~~~~~&4 E ~~~~~~~~+ 49 M +3 / +29 I "NSepY FIG. 4. Effect of spacing alteration mtations on rrna transcription. (A) Rnoff analysis of linker scanning mtations that position the pstream element closer to or frther from the core element and corresponding 5' deletion mtations. Two linker scanning mtations (LS-130/-120 and LS-137/-131) which inactivate the pstream element were also inclded for comparison. A wild-type control gene (WT) was inclded in each reaction. (B) Diagram showing the position of the pstream element in each mtant and the transcription level relative to that of a wild-type gene. Transcription efficiency was measred as described in the legend to Fig. 2. The small nmbers above each slash (which show the approximate position of the linker) indicate the nmber of ncleotides wich were deleted or inserted in each mtant. CO ( nated the core control element (-45 to +18) and the pstream control element (-156 to -107). These two elements are fnctionally noneqivalent and share no homology in seqence. The core element appears to be an essential signal for RNA polymerase I transcription becase most mtations in this region cased a 5- to -fold redction in the level of transcription. The nearly complete lack of homology in the core region of different mammalian RNA polymerase I promoters is consistent with the idea that these seqences may also play a major role in the species-specific recognition by RNA polymerase I. A role for the core element in species specificity is in fact sggested by the finding that mtations in this element mimic the effects observed when the speciesspecific transcription factor, S, is removed from the transcription reaction (12). There is a small stretch of homology (+4 to +16) in the core region between the hman and mose promoters. This short conserved segment, however, appears to play a relatively minor role in determining promoter efficiency in vitro and in transient expression experiments becase base sbstittion or deletion of these seqences redced transcription only by a factor of twofold. Nevertheless, these experiments do not rle ot the possibility that this conserved segment is of greater importance nder certain physiological conditions in the cell. Analysis of altered templates that contained two tandemly arranged core elements indicates that the presence of a second element actally inhibits transcription. This reslt sggests that the interaction of the transcriptional machinery

7 VOL. 6, 1986 PROMOTER ELEMENTS IN HUMAN rrna GENE 233 A 4) 'I -.II I 4B B 1343 _ I l IN l v,, U S-_i'. _ ~~~~1048- _ W -- ~~~766 _ ~~543_ C WILD-TYPE , ~~~~~~~~~~i......i i 11 JRSRASCRBP JIf0 essssr AZA LS-107/-156 / DIS- 186/-1} / UCE LS 1 0/-45 --O- +11 _/I /--Emm./.. WT--bE O- --Emm --pp- LS-24/-45 _ / FIG. 5. Effect on transcription of dplication or inversion of an element. (A) Rnoff analysis of linker scanning mtations (LS) that dplicate the pstream element, contain two linkers, or change the orientation of the pstream element. A wild-type control gene (WT) is inclded in each reaction. (B) Rnoff anlaysis of two linker scanning mtations that dplicate the core element. The transcripts derived from the two initiation sites are indicated. M, Size markers (ncleotides). (C) Diagram showing the nmber and orientation of both control elements in each mtant and their relative transcription levels. Transcription efficiency was measred as described in the legend to Fig. 2. The slashes indicate the approximate position of the linker and the nmber of ncleotides inserted. The arrows indicate the orientation of the pstream element relative to the core element. fn ;10 C,. _~~ W YWT -_ii _p~ Ml.,.t 1t t FIG. 6. Primer extension analysis of RNA transcripts derived from mtants that alter the spacing, mltiplicity, or orientation of the pstream element. The expected positions of the cdna prodcts are shown for the mtants (LS) and the psedo-wild-type gene (TWT). AG, G, Maxam and Gilbert seqencing markers.

8 234 HALTINER ET AL. WT5-.. A 5 : L...I I I 1. II I.-1 FIG. 7. Variable effects of pstream seqences on rrna transcription. Rnoff analysis of transcripts derived from a wild-type gene (WT) and A5-83 (A5). The transcription reactions contained 125 ng of template (lanes 1, 4, and 7), 250 ng of template (lanes 2, 5, 8, and 10), or 500 ng of template (lanes 3, 6, and 9). The amont of extract sed was 150 ±Lg (lanes 1 throgh 3 and 10), 225,g (lanes 4 throgh 6), or 300,g (lanes 7 throgh 9). The reaction in lane 10 was spplemented with 50 ng of a heparin-agarose 0.5 to 1 M NaCl steps (SL1) and 24 p.g of a heparin-agarose 0.2 to 0.4 M NaCl step. 10 with the core seqence redces the accessibility of nearby adjacent core seqences in these doble core mtants. These experiments confirm that all the seqences necessary for accrate RNA polymerase I transcription initiation are contained within seqences from -45 to + 10 relative to the start site. Ths, the core element differs from the pstream element in its qalitative as well as its qantitative effect on transcription, since it determines the transcription start site. The pstream control element incldes a 21-bp stretch of DNA that is highly conserved between hman, mose, and rat DNas; this element plays an important role in modlating the efficiency of transcription (approximately fivefold) bt is not absoltely reqired for transcription in vitro from the hman ribosomal promoter. The transcription phenotypes of the spacing and inversion mtations indicate that the role of the pstream control element depends on the core element in a manner which is both directional and distance dependent. The importance of the pstream element for transcription was first observed in in vitro reactions with deletion mtants, bt recently it has also been demonstrated by an in vivo transfection assay (29). The transfection assays frther revealed that an additional far-pstream seqence located from -234 to -167 relative to the RNA start site may also contribte to transcription efficiency of the RNA polymerase I promoter. This far-pstream region stimlates transcription approximately fivefold, making the total stimlatory effect of seqences pstream of the core element 25-fold on transcription in vivo. Similarly, we observed that the presence of pstream seqences can stimlate transcription 50-fold in vitro when the reaction is spplemented with fractionated transcription components. Whether this additional stimlation is de to the far-pstream control elements observed in vivo will reqire frther analysis. Taken together, these observations sggest that seqences pstream of the core element play an important role in activating RNA polymerase I transcription and that this effect is likely to be mediated by trans-acting protein factors that interact with these control seqences. RNA polymerase I promoters have been mapped in several other systems by deletion analysis and point mtagenesis in vitro. In the case of the mose promoter, an element which extends from approximately -35 to +9 (especially ncleotides -7, -15, and -16) has been shown to have a sbstantial effect on transcription in vitro, and seqences pstream of -45 affect transcription in competition experiments (4, 10, 28, 33). Recently, the mose pstream element has been mapped in greater detail to a position similar to that of the hman pstream element (21). However, the mose pstream element appears to have a somewhat lesser effect on transcription in vitro, and this effect is observed only nder sboptimal transcription conditions with high salt concentrations, pstream trncated templates, or spplementing the S- extract with specific phosphocelllose chromatographic fractions. The rrna promoter of Xenops laevis also appears to extend from -142 to +6, and its effect on in vitro transcription varies depending on the conditions sed (30). Unlike mammalian RNA polymerase I, however, Xenops transcription appears to be stimlated in vivo nder certain template competition conditions by the presence of seqences located several kilobases pstream from the initiation site (11). The RNA polymerase I pstream element shares some interesting similarities and differences when compared with previosly characterized RNA polymerase II promoter elements. For example, like the simian virs base repeat element (1) and the herpesvirs thymidine kinase distal elements (18), the rrna promoter pstream element is positioned in the nontranscribed region 5' to the initiation site and affects the efficiency rather than the accracy of transcription. However, several factors distingish these RNA polymerase II pstream elements from the RNA polymerase I element. First, the RNA polymerase II elements appear to fnction in either orientation with respect to the start site of transcription (3, 19), while the RNA polymerase I pstream element does not. Second, the mechanism of RNA polymerase II activation has been shown in several cases to involve the binding of seqence-specific DNA binding proteins to the pstream elements (2, 8, 9a, 25). In the case of RNA polymerase I transcription, no sch binding has been detected with the highly prified transcription factor, SL1. These differences in pstream elements of the RNA polymerase I promoter and RNA polymerase II promoter sggest a fndamental difference in the mechanism of activation of transcription by these two systems. ACKNOWLEDGMENTS MOL. CELL. BIOL. We thank K. Jones, C. Kane, R. M. Learned, S. Mansor, and D. Rio for critical reading of the manscript and also K. Ronan for typing the manscript and J. Smith for assistance with the figres. This work was spported by Pblic Health Service grant GM from the National Instittes of Health and partially fnded by a National Institte of Environmental Health Sciences Center grant. LITERATURE CITED 1. Benoist, C., and P. Chambon The SV40 early promoter region: seqence reqirements in vivo. Natre (ndon) 290: Dynan, W. S., and R. T. Tjian The promter-specific transcription factor Spl binds to pstream seqences in the SV40 early promoter. Cell 35: Everett, R. D., D. Baty, and P. Chambon The repeated GC-rich motifs pstream from the TATA box are important elements of the SV40 early promoter. Ncleic Acids Res. 11: Grmmt, I Ncleotide seqence reqirements for specific initiation of transcription by RNA polymerase I. Proc. Natl.Acad. Sci. USA 79: Grmmt, I., E. Roth, and M. Pale Ribosomal RNA transcription in vitro is species specific. Natre (ndon) 296: Grmmt, I., V. A. Smith, and F. Grmmt Amino acid starvation affects the initiation freqency of ncleolar RNA

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