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Mol. Cells, Vol. 3, pp. 133-136 Cooperative Function of Upstream and Core Domains of the Yeast Ribosomal RNA Gene Promoter Jae Kyun Rho, Hyuk Ran Kwon, Ronald H. Reeder l and Soo Young Choe* Department of Biology, Chungbuk National University, Cheongju 360-763, Korea; Ifiutchinson Cancer, Research Center, Seattle, WA 98104, USA. (Received on February 27, 1993) We have previously reported that the yeast, Saccharomyces cerevisiae, ribosomal gene promoter contains at least two essential domains, an upstream domain located at the 5' boundary near position - 150 and a core domain around the site of transcription initiation at + 1, by in vitro analysis using whole cell yeast extracts. Here we show the activity of the promoter is highly sensitive to spacing changes between two domains, but the activity can be partially rescued when the spacing changes was either increased or decreased to 10 bp. The upstream or core domain DNA sequences alone could not compete with wild promoter in transcription initiation step, and the upstream and core domains are both necessary for transcription initiation complex formation. The upstream domain of the promoter, however, can be severed from the core promoter domain once the stable complex has been formed. These results suggest that the yeast ribosomal gene promoter has a critical requirement for binding of protein or protein complex to core and upstream domains to be located at precise positions on the face of the DNA helix. Saccharomyces cerevrszae ribosomal RNA genes, which code for 35S rRNA precursor, are known to contain two distinct DNA elements (diagrammed in Fig. 1) which influence transcription initiation by RNA polymerase I. One of these elements is the gene promoter situated at the 5' end of the 35S coding region. Analysis of the gene promoter both in vivo (Musters et al., 1989) and in vitro (Kulkens et aI., 1991 ; Choe et al., 1992) shows that it consists of about 150 bp of sequence which slightly overlaps the site of transcription initiation and that it has a structure similar to that of vertebrate ribosomal gene promoters. The other DNA element is the ribosomal gene enhancer. The enhancer was originally described as a 180 bp EcoRl to HindIII fragment that is located at the 3' end of the 35S coding region by in vivo studies (Elion and Warner, 1984, 1986). Stewart and Roeder (1989), however, found that another critical region for the transcription enhancement, just to the right of the HindIII site. We have recently reported a simple procedure for obtaining whole cell extracts from yeast that are active for polymerase I transcription as well as for transcription by polymerase II and III (Schultz et al., 1991). Using this extract we also showed that the ribosomal RNA gene promoter can be separated into core and upstream domains (Choe et aI., 1992). Yeast and vertebrate promoters are of similar size and both can be separated into core and upstream domains. In this paper we show that these two domains seem to be necessary for positioning of transcription initiation factors on the correct face of the DNA and to be con- * To whom correspondence should be addressed. nected via transcription initiation protein or transcription initiation protein complex. Materials and Methods Plasmid constructs pYrllA was the parent plasmid for all constructs. It contains the 35S ribosomal gene promoter isolated as a Smal-Taql fragment (- 216 to + 25) from pBD4 (Bell et al., 1977). The fragment was modified by ligating a 16 base pairs (bp) Xhol linker to the Taql site at +25 and then it was cloned into the large HincIIXhoI fragment of a pGEM3 derivative (pGEM3-EX). pYr12-5 is the same as pYrllA except that a 26 bp Xhol linker was inserted into the Taql site at +25 (Schultz et al., 1991, Choe et al., 1992). Novel Xbal sites were introduced into the promoter by oligonucleotide-directed mutagenesis (Kunkel, 1985). Spacing changes within the gene promoter were constructed at the positions between -125 and - 105. A 20 bp deletion mutant was made by digesting -129 / -102 linker scanner mutant with Xbal and religating. A 16 bp deletion mutant was made by digesting - 129 / - 102 linker scanner mutant with Xbal, ftlling the sticky ends, and religating. All other spacing mutants were made by cutting 20 bp deletion mutant between - 125/ -105 with Xbal and inserting the appropriate size of double stranded oligonucleotide (sequences of the final constructs are shown in Fig. 2A). All mutations were verified by direct sequencing of the final construct. In vitro transcription Extracts (100,000 X g supernatants) were prepared © 1993 The Korean Society of Molecular Biology according to Schultz et al. (1991) from cells grown in YEPD to OD600 1-4 and broken with a mortar and pestle under liquid nitrogen. Transcription reactions were performed at 22 °C as described (Choe et aI., 1992), except that they were in 20 Iil, contained 90 mM KC1, and the nucleotides were at 500 f.LM each. All reactions in a series received the same amount of extract, in the range of 40-80 Ilg protein, and were run for 30-60 min. Assay of RNA polymerase I transcnptzon Sl nuclease protection assays were performed essentially according to Labhart and Reeder (1986), using single-stranded DNA probes. The Sl nuclease protection probe for transcription of the pYr11A was a 50nucleotide ·single-stranded oligonucleotide complementary to pYrllA from -15 to +35. The Sl probe for detecting transcripts from pYr12-5 is 60-nucleotide single-stranded oligonucleotide complementary from -15 to +45. Results and Discussion Spacing between two domains gives a partial rescue of promoter activity All of the work in this paper was done with a promoter fragment which extends from an SmaI site at position -216 upstream of transcription initiation to a Taq I site at position + 25 downstream of initiation. At the TaqI site a 16 or 26 bp linker has been inserted to facilitate distinguishing transcripts from this promoter from endogenous transcripts. The sequence of this fragment is shown in figure 1. To examine the possible' role of domain spacing in the yeast ribosomal gene promoter we made various push _apart and pull together spacing mutants. As shown in Figure 2 we first removed 20 bp between the position of - 125 and -105. Secondly, we inserted additional nucleotides to create push apart mutants. The transcription activity of both the pull together and push apart spacing mutants is shown in Figure 2B. Changing the spacing .at this location is very dele- terious to promoter actIvIty. For example, either increasing or decreasing the spacing by 5 bp causes activity to drop to about 10% of wild type. The most interesting result, however, is that a partial rescue of promoter activity is observed when the spacing is further increased or decreased to 10 bp. This type of periodic dependence on spacing has been previously described for domains of the Xenopus [aevis ribosomal gene promoter (McStay and Reeder, 1990; Pape et aI., 1990). . We interpret these results to me~ n that spacing between the core and upstream domains of yeast promoter is critical. Furthermore, transcription factors bound to the core and upstream domains of the yeast promoter must be positioned on the correct face of DNA for maximal activity. The upstream domain is dispensable after formation of stable transcription initiation complex As shown in Figure 2, precise spacing between these two domains is essential for promoter activity but much of the sequence betWeen the two domains can be replaced with little effect as long as the original spacing is maintained. Covalent linkage between the two domains appears to be essential to allow stable promoter complex formation. We wondered whether covalent linkage was still essential after the stable complex had been formed. To answer this question a promoter was incubated in extract, in the absence of triphosphates, to allow stable complex formation. Then a restriction enzyme was added to cut the pro- A ·1 16 -100 I I ~ I I -- - -TTTCTCGGCAAGAAATACGTAGTTAAGGCCGAGCGACAGAGAGGGCAAA---- I tct--------------------aga tctagct----------------aga tctagagcggtct----------aga tctagagcatggcggtct-----aga tctagacgccggcatggcggtctaga tctagacgccggcatggcggtctrga gctct I -20 · 16 · 10 ·S +S + J() ' IS + 2U tctagacgccggcatggcggtct~rg=--a_-,-..., gcaccagtct tctagaggcagctggtgcctaccf-'rg'---a--:-_ _-,---, gccatgccggcgtct tctagagccctggcagctggtgcaga I B • ctaccgccatgccggcgtct , CCCGGGCCACCTCTCAc.TTTCCAA.AAAAATATACCCTMGATTTTTCC.AGAATACCTT , -IJU Core domain A .1)0 Mol. Cells Yeast Ribosomal RNA Gene Promoter 134 , s-J .UXI AAATTCAAGTTTrTCTCCGCMCAAATACGTACTTAAa:x:J:.CACCCN:ACAGAcaxAAAACAAAATAM B ·20 ·16 ·I ll.~ 0 .., 0 ., - 10 'I ~ ·2CJ , .jO AGTAAGATITTAG1TTCT AA T'GCCACGGGGGCiiTAGTCATCGACTACAACTCl'CACCAAMCTAcrrcc ,.1 ( 16 bp CAGCTACTTCATCCGAAAGCAClTGAAGACMCITCCTCMCACCCT'CCAG rr.. o linl xw Transcription signal CCI<JTCTCACCCTCAAGAcccrCGAC ( 26 bp Iinl "'" Figure 1. (A) Diagram of the yeast ribosomal gene and its spacer region. (B) Sequences of the ribosomal gene promoter. The promoter region was subcloned and tagged by inserting an XhoI linker (underlined) into the TaqI site 23 bp downstream from the transcription start site. Figure 2. Effect of changing the spacing between upstream and core promoter domains. (A) Sequence of spacing mutants in -125/ - 105. Dotted line depicts the deleted sequences. (B) Transcription activity from the - 125/ -105 spacing mutants. ----- - ••• I 2 3 4 A 5 6 7 8 • 9 10' 11 12 A • .2 Seal 1-------f:,/~ Seal LS-1 2'J/-I02 1 Seal LS-I O'J/- I02 1 7 8 9 .2 I .2 L.....J I .2 L.....J I 0 pmole L.....J -2 16/- 103 -21 6/-83 -2 16/-63 -21 6/-43 B Xbal ", ,Xbal ,~ _ 3 4 5 6 7 8 9 10 II 12 Transcription signal Seal Figure 4. Competition effect of the promoter fragments in transcription. (A) 3' deleted promoter fragments were used for competition. In case of lanes I, 3, 5, 7, 0.2 pmole DNA was used for competition and in lanes 2, 4, 6, 8, one pmole was used. Lane 9 shows the transcription signal in the absence of competitor. (B) The plasmids which contain the 5' deletion promoters were used for competition. The final concentration of the competitor plasmids were 20 Ilg/ml. ----11- - - 1 Xbal ,~ ' Seal /l-----i _ ,'/----r-= 2 -1 55/+35 -1 02/+35 -42/+35 pGEM -2 16/+35 -142/+35 -82/+35 -22/+35 ·1 62/+ 35 -1 22/+ 35 -62/+3 5 -2/+35 ,~ __ I Template Xbal digestion Xbal XbaI 6 4 Transcription signal - +++ II---.ic_ I L.....J Same as 1-4 Same as 1-4 p YR I I A 3 Transcription signal pYrilA LS-129/-102 LS-I09/-102 - +++ 2 Template • • B C 135 Jae Kyun Rho et at. Vol. 3 (1993) Seal _ - - - - 11- - - 1 Figure 3, The upstream element of the promoter can be severed from the core region, following stable complex formation. (A) Demonstration that XbaI completely digests templates in the presence of whole cell extract Constructs were cut with Seal, end-labeled with 32p , put through the reaction protocol up to the point of adding nucleotide, at which point nucleic acids were isolated for electrophoresis. (B) Transcription results. Lanes I, 5, 9, initiation signal from three different promoter mutants. Lanes 2, 6, 10, addition of Xbal prior to stable complex formation. Note that pYrilA does not contain an XbaI site. Lanes 3, 7, II , addition of heat inactivated XbaI after stable complex formation. Lanes 4, 8, 12, addition of active Xbal after stable complex formation, prior to addition of nucleotides. (C) Diagram of the template DNA moter between the core and upstream domains. Finally, triphosphates were added and transcription capacity was measured. Figure 3B shows the results of this type of experiment as perfonned on three different variants of the promoter. The first promoter tested was a wild type promoter (pYrllA) which does not contain an XbaI site between two domains (Fig. 3C). Addition of the enzyme XbaI, either before stable complex formation (lane2) or after complex fonnation (lane 4) had no effect dn transcription. The same experiment was then perfonned using a promoter containing a large substitution of foreign sequence between positions - 129 and - 102. An XbaI restriction site is present at either end of this substitution (Fig. 3C). As we have shown previously, this sub- stitution mutation is relatively neutral when the intact promoter is assayed (Choe et al., 1992). If this template is digested with XbaI prior to stable complex fonnation, transcription is nearly eliminated as expected (7% of control, lane 6 in Fig. 3B). However, if XbaI digested is perfonned after · stable complex fonnation, about 31 % of the control activity remains (Fig. 3B, lane 8). Figure 3A, lane 8, shows that digestion was complete in this particular reaction, even in the presence of the prefonned stable complex. We repeated the experiment with another mutated promoter, LS-I09/-1Q2, which contains a single XbaI site at the indicated location between the upstream and core domains (Fig. 3C). Cutting at this site prior to stable complex fonnation also destroys promoter activity (4% of control, Fig. 3B, lane 10). However, complete cutting after stable complex fonnation still allows about 58% of the control activity (Fig. 3B, lane 12). This strongly suggests that the upstream domain of the promoter is essential for assembly of the stable promoter complex but physical linkage is not needed after that assembly has occurred. Both upstream and core domais are necessary for competing against wild type promoter The promoter can be divided into two domains and the upstream domain is proved to be necessary for transcription as shown above (Fig. 3B, lane 6 and lane 10). In general it is known that the promoter sequences serve as the binding site for specific tra'n scription factor proteins. If there is a transcription factor, which can bind only to the upstream domain, the transcription activity of the promoter should be decreased in the presence of excess amounts of upstream DNA. 136 Yeast Ribosomal RNA Gene Promoter Figure 4 shows the results of competetion experiments. We used two different sets of DNA fragments for the competition assay. All DNA fragments of one set have upstream domain of the promoter and various downstream length of promoter. For example, all the four DNA fragments in Figure 4A have the same 5' boundary of the gene promoter and have various length of internal promoter sequences. Only the fragment of -216/ -43 covers both of the upstream and core domains. We preincubated these fragments with yeast cell extracts for 10 min at room temperature in order to form the DNA-protein complex and then added the template DNA for the transcription. Interestingly all the fragments, except -216/- 43, could not compete against the promoter (Fig. 4A). When the fragment - 216/- 43 is used as the competitor, the transcription from the intact promoter is strongly inhibited. This result shows that some protein(s) only can bind to the upstream DNA at the presence of core domain DNA We also used the plasmids which contain core domain DNA and various length of upstream DNA as the competitor against the promoter activity (Fig. 4B). As expected the transcription was strongly inhibited by the DNAs which contain both upstream and core domains (lane 2-5). The core domain DNA, however, still showed some transcription inhibitory effects, and the transcription signals were basal level (lane 6-9). These results strongly suggested two things. One is that the core domain DNA could serve as the first binding site for some protein(s), and another is that the upstream DNA binding protein(s) could bind to the upstream domain after formation of core DNAprotein complex. Mol. Cells Acknowledgments This work was supported by a Genetic Engineering Research grant from Ministry of Education, 1991. References Bell, G. I., DeGennaro, L. J., Gelfand, D. H., Bishop, R J., Valenzuela, P., and Rutter, W. J. (1977) J Bioi. Chern. 252, 8118-8125 Choe, S. Y., Schultz, M. c., and Reeder, R H. (1992) Nucleic Acids Res. 20, 279-285 E1ion, E. A , and Warner, J. R (1984) Cell 39, 663-659 Elion, E. A , and Warner, J. R (1986) Mol. Cell. Bioi. 6, 2089-2097 Kulkens, R , Riggs, D. L., Heck, J. D., P1anta, R J., and Nomura, M. (1991) Nucleic Acids Res. 19, 53635370 Kunkel, T. A (1985) Proc. Nat!. Acad. Sci. USA 82, 488- 492 Labhart, P., and Reeder, R H. (1986) Cell 37, 285-289 McStay, B., and Reeder, R H. (1990) Genes and Dev. 4, 1240-1252 Musters, W., Knol, J., Maas, P., Dekker, A F., van Heerikhuizen, H ., and P1anta, R J. (1989) Nucleic Acids Res. 17, 9661-9678 Pape, L. K , Windle, J. J., and Sollner-Webb, B. (1990) Genes and Dev. 4, 52-62 Schultz, M. c., Choe, S. Y., and Reeder, R H. (1991) Proc. Natl. Acad. Sci. USA 88, 1004-1008 Stewart, S. E., and Roeder, G. S. (1989) Mol. Cell. Bioi. 9, 3463-3472