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Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8557-8561, December 1985 Genetics The relationship between the "TATA" sequence and initiation sites at the HIS4 of (yeast/mRNA/gene regulation) FuMIKIYo NAGAWA* AND GERALD R. FINKt tWhitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; and *Wakunaga Pharmaceutical Company, Ltd., 1624 Shimokodach: Kodacho, Takadagun Hiroshima, Japan Contributed by Gerald R. Fink, July 25, 1985

ABSTRACT Transcription of the HIS4 gene begins at a Second, there are often multiple TATA sequences because single site (I) at position -60 from the ATG that begins the 5' noncoding regions of yeast are very T+A-rich. . We have made linker insertions/deletions in the 5' Third, some yeast genes have a single major transcript (15, noncoding region to identify the elements required for the 17-20), whereas others have multiple transcripts spread over specificity of transcription initiation. Although there are four a 100-bp region (20, 21). sequences that begin TATA and are near the start of tran- The HIS4 gene is particularly useful for a study of the scription (-170, -132, -123, and -102) only the sequence at factors influencing transcription initiation in yeast. The HIS4 -123 (TATA-123) is required for transcription initiation. By gene specifies a single transcript even though there are inserting synthetic oligonucleotides into a mutant from which several TATA sequences upstream from the site oftranscrip- TATA-123 had been deleted, we found that just TATA or tion initiation (22). This transcript is regulated by short TATAA does not work but that TATAAA functions almost as repeated sequences (called UAS, upstream activation sites) well as the wild-type sequence. This hexamer does not work in upstream from all the TATA boxes (23, 24). The HIS4 repeats the opposite orientation (TTTATA). When a synthetic TATA are typical yeast UAS sequences: they regulate transcription sequence is placed upstream from the normal site, the site of in both orientations and at variable distances from the start initiation also moves upstream in a roughly cometric way even of transcription (25). In this report we examine sequence when TATA-123 is present. Analysis of transcripts in strains requirements for the TATA element and the relationship where the distance between the TATA sequence and the between the position of the TATA element and the site of wild-type site oftranscription initiation (I site) has been altered transcription initiation. shows that in yeast, unlike higher cells, transcription does not initiate at a strictly dermed distance from the TATA sequence. Constructions that alter the distance between the TATA and MATERIALS AND METHODS the I site or remove the I site change the pattern oftranscription Strains. Yeast strain TD28 (MAT-a ura3-52 inol) was used initiation without affecting the level of HIS4 expression. Dele- as the yeast host for analysis of RNA and linker insertions. tions that eliminate the I site produce heterogeneous transcripts Escherichia coli HB101 was used for preparing plasmid and deletions that substantially shorten the distance between . TATA-123 and the I site initiate at multiple sites downstream Construction ofPlasmids. The plasmid pFN7, used to make from the I site. Thus, both the TATA and the sequences linker insertions in HIS4, was made by ligating together a downstream from it determine the pattern of transcription Sau3A fragment containing the 5' noncoding region of HIS4 initiation. (22) and the EcoRI-Nde I fragment of pBR322 after each fragment was filled in with the Klenow fragment of E. coli Eukaryotic promoters recognized by RNA polymerase II are DNA polmerase I. Linker insertions were made as described divided into several functionally distinct regions: the initia- (24), and each plasmid containing an was designated tion site (I), the "TATA box," and the upstream regulatory pFN7x-n where n is the mutant isolation number. The elements. These elements are found in the 5' position of each linker insertion was obtained from the untranslated region adjacent to the structural gene (1-8). The sequence (26). TATA box appears to be a sequence required in most Plasmid pFN8 was made by ligating a BamHI-Sal I mammalian promoters for efficient initiation of transcription fragment containing E. coli lacZ from YIp334 (23) with the (4, 5, 9). Transcription usually initiates at a site 30 base pairs BamHI-Sal I fragment from pMR79, a Ycp50 derivative (bp) downstream from the TATA box, which suggests a lacking a Xho I site (a gift ofM. Rose). The resulting plasmid, spatial relationship between the TATA box and the actual site pFN8, was used to construct lacZ fusions with each of the of transcription initiation (I site). In , there is no linker-insertion mutants. To fuse the linker-insertion muta- evidence for specific sequence requirements at the I site; its tions to lacZ, we cut plasmid pFN8 with HindIII, filled in the location is determined by the position ofthe TATA box (1, 10, ends with Klenow fragment, cut with BamHI, and then 11). separated the fragments by electrophoresis in an agarose gel. In yeast, sequences similar to the TATA box have been This lacZ fragment was ligated with the Xmn I-Sau3A found upstream of many structural genes (12, 13). However, fragment from each ofthe linker-insertion mutants (pFN7x-n) it has been difficult to identify the exact sequence that so that the HIS4 5' region containing the linker was joined functions as a TATA box simply by inspection. First, the in-frame to lacZ at the Sau3A site. The structure of each distance of these sequences from the start of transcription fusion plasmid was confirmed by restriction analysis. These differs from gene to gene. In some cases the first TATA is lacZ fusion plasmids containing Xho I linker insertions are more than 100 bp from the start of transcription (14-17). called pFN8x-n. All linker and oligonucleotide insertions de- The publication costs of this article were defrayed in part by page charge scribed in this study were sequenced to give exact informa- payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: UAS, upstream activation site(s); bp, (s). 8557 Downloaded by guest on September 24, 2021 8558 Genetics: Nagawa and Fink Proc. Natl. Acad. Sci. USA 82 (1985) tion on the location, orientation, and copy number (26). In the Table 1. ,B Galactosidase activities in insertion mutants experiments represented in Fig. 4 the oligonucleotide TATATAATA was inserted into the Xho I site of plasmids Activity pFN7x-101, -170, -210, -407, and -99 (see Fig. 1). Those Mutant R D recombinants containing a single copy of the oligonucleotide pFN6 (wild type) 417 1380 in the correct orientation were cut with Pst I and Acc I and pFN8 (no promoter) <1 <1 ligated to the Pst I-Acc I fragment obtained either from wild x-52 (-316, -144) 1 1 type or from TATA x-172 (see Fig. 1). Those x-203 (-306, -207) 8 543 recombinants having the TATATAATA oligonucleotide x-99 (-286, -261) 322 1109 joined either to a fragment with the wild-type TATA or to a x-207 (-265, -242) 13 548 fragment from x-172 were fused to lacZ as described for the x-114 (-237, -213) 12 580 pFN8x constructions. x-408 (-226, -213) 92 1231 Assay for .8-Galactosidase Activity and RNA. Transform- x-407 (-217, -210) 501 1485 ants were grown to mid-logarithmic phase in minimal medium x-406 (-217, -203) 329 1186 SD (27), for repression, and for 6-8 hr in SD plus 10 AuM x-402 (-217, -181) 94 197 3-aminotriazole, for derepression (28). The assay for j3- x-18 (-217, -172) 36 100 galactosidase was conducted as described (24). Preparation x-1 (-202, -181) 225 421 of RNA and analysis of the 5' ends by primer extension was x-401 (-202, -172) 323 443 carried out as described by Teem and Rosbash (29) except x-210 (-192, -177) 320 1076 that the reaction mixture contained 6.5 jig of total RNA and x-170 (-184, -162) 415 1305 was incubated at 30'C with avian myeloblastosis re- x-101 (- 156, -133) 620 1235 verse transcriptase. The M13 sequencing primer (15-mer, x-122 (-133, -108) 65 76 New England BioLabs) was used because this primer has x-204 (-130, -106) 79 124 homology to the lacZ coding region and the 5' end of this x-172 (-126, -101) 36 30 primer is located about 25 bp downstream from the fusion x-46 (-119, -96) 30 38 point of HIS4 and lacZ. The primer-extended DNA was x-44 (-114, -86) 336 890 analyzed on an 8% acrylamide sequencing gel. x-131 (-103, -82) 312 919 x-7 (-102, -94) 337 1331 x-176 (-72, -49) 305 1229 RESULTS x-33 (-72, -44) 316 797 The TATA at -123 (TATA 123) Is Required for Transcrip- x-198 (-36, -14) 296 919 tion Initiation. We used linker-insertion/deletion mutations Cells were grown and assayed as described in Materials and to determine the sites important for the transcription ofHIS4. Methods. R, repressed; D, derepressed. The numbers in parentheses There are four sequences that begin TATA and are near the are the endpoints of the deletions (see Fig. 1). pFN6 is a HIS4-lacZ start of transcription. These are located -170 (TATAGAA), fusion plasmid with no insertion/deletion in HIS4 (wild type), and -132 (TATACTG), -123 (TATATAA), and -102 bp pFN8 is a plasmid containing a lacZ region devoid of a functional (TATATTC) from the ATG translation start ofHIS4 (Fig. 1). promoter. Deletions of the TATA sequence at position -102 (x-44, x-131, x-7) fail to affect the level of HIS4 expression or both the -102 sequence and the -123 sequence (x-172, x-46), regulation (Table 1). Deletions that lack the -170 TATA or -123 and the -132 sequence (x-122, x-204) produce very sequence (x-20, x-170, x-31, x-130) also produce almost low levels of,-galactosidase activity and this activity appears normal levels of /3-galactosidase. However, mutants lacking to be unregulated. These results suggest that of the four

- 300 - 250 -200 -150 -100 -50 +I bp I ~~~~~~~~~~~~~~I ~ ~ ~ TATA I ATG 5 2

152 '- 59 - 311- 99- 130 37 101 207'- 1221 204 '--- 172II' 408- 191-' :44:1- 407'1- 131 4061 71- 402 176 - 33' 1981 ~~~~~~~1.4011- 95 i-l 210 841-4 20! ' 70

FIG. 1. Map of the linker-insertion/deletion mutants. The line at the top represents the HIS4 5' noncoding region. The numbered bars beneath this line show the extent of each deletion. Each mutant has a Xho I linker at the site of the deletion. Open arrows represent the HIS4 regulatory repeats (TGACTC). Open boxes indicate the location of TATA sequences, and the black box represents TATA-123. I, the site of transcription initiation in the wild type. The hatched box represents the ATG initiation codon of the HIS4 gene. All of the constructions are joined to lacZ at a Sau3A site 30 bp from the A of the ATG. Downloaded by guest on September 24, 2021 Genetics: Nagawa and Fink Proc. Natl. Acad. Sci. USA 82 (1985) 8559 TATA sequences only the one at -123 is required for normal x-131 is a larger deletion in the same region (Fig. 1) that brings expression of HIS4. the TATA sequence and the I site 12 bp closer than they are Deletions of TATA-123 (x-46, x-172, x-204, and x-122) in the wild type. This deletion alters both the position of the result in failure to initiate at or near the site (I, at -60) of major initiation site and the number ofinitiation sites; a major wild-type initiation (see Fig. 3). Strains carrying these dele- transcript in x-131 starts 11 bp 3' of the normal I site, and a tions produce several regulated but nonfunctional transcripts minor transcript initiates at the I site (Fig. 2). Deletion x-44 that initiate downstream from the ATG within the coding deletes 19 bp between TATA and the I site and the major sequence of HIS4. There are a number of potential TATA transcript initiates at the same place as that in x-131. sequences between the I site and the ATG that could be However, in strains carrying the larger deletion (x-44), the responsible for these short, nonfunctional transcripts. amount of the major transcript 3' of the I site is reduced and The Function of the I Site. Deletions that remove the I site there is almost no detectable transcription originating from have profound effects on the site ofinitiation and multiplicity the I site. These data suggest that the spacing between the oftranscripts but no effect on the level ofHIS4 expression or TATA sequence and the I site is important. In fact, the regulation (Table 1). Deletions x-176 and x-33 eliminate the normal I site is not recognized when it is too close (44 bp) to wildtype transcription initiation site (I) and produce a TATA-123- collection ofheterogeneous transcripts. The major transcript Deletions on the 5' Side of TATA-123. Insertion/deletion in each deletion begins 58 bp from the first base ofTATA-123. mutations on the 5' side ofTATA_123 do not affect either the The rest of the transcripts start at similar sites in both site of initiation of the HIS4 transcript or the multiplicity of deletions. termini (Figs. 1 and 3). Deletions with fewer than three UAS We examined deletions between TATA-123 and the I site repeats also result in initiation at the I site, whereas those that (Figs. 1 and 2) to determine the effects of altered spacing on have lost all three repeats abolish transcription completely the site of initiation of transcription. Three of the mutants (data not shown). Deletions that remove two of the three contain deletions of different sizes but retain TATA-123 and repeats and some of the deletions in the region between the the wild-type I site. The x-7 results from a 7-bp two upstream repeats lower the basal level of expression deletion and an 8-bp linker insertion making the distance (x-114, x-97, x-19, and x-408; Fig. 1 and Table 1). Deletions between TATA-123 and the I site longer than wild type by 1 ofthe third repeat (x-1 and x-401) affect the derepressed level bp. Strains carrying x-7 initiate the transcript at the same site and not the basal level of expression. Despite the fact that with these upstream deletions alter the level of expression or (I) and the same efficiency as the wild type. Mutation change the magnitude of derepression, all result in initiation at the I site. CU CMt C) N c N cm The Sequence of Functional TATA Elements. We deter- WTMDRCOXc - --_D mined the minimal sequence required for proper initiation at MR DR DRDRDR D RDRDR DRDR DR D the -123 site by inserting synthetic oligonucleotides into the Xho I site of deletion x-172 (Fig. 1). Insertion of oligonucleo- tides containing the same sequence found in wild type (TATATAATA) produces levels of 3-galactosidase activity indistinguishable from that produced by the wild type (Fig. 3). In addition, the oligonucleotide sequence TATAAA CD functions almost as well as the wild-type sequence but does not function when placed in the opposite orientation (TT- TATA). Oligonucleotides containing just the sequence 1+ 1- 2+ 2- 3+ 4+ 4- 5+ 5- WT m m-11 -I11 R DR D R D R D R D R DR D R D R D R D M

FIG. 3. Transcription of HIS4 in strains containing synthetic TATA sequences. Total RNA was prepared from cells and was characterized as described for Fig. 2. Numbers above the brackets FIG. 2. HIS4 transcription in strains carrying linker mutations. stand for the oligonucleotide inserted (see below); + and - indicate The 5' ends of messages in these strains (see Fig. 1 for the extent of the right and wrong orientation (i.e., 5- is 'ITTATA). WT, wild type; each deletion) were mapped by primer extension. Total RNA was R, repressed; D, derepressed. M, markers (pBR322 cut with Hpa I). prepared from cells grown under repressing (R) and derepressing (D) Each of the oligonucleotides has Xho I cohesive ends and was conditions. Primer extension was conducted as described in Mate- inserted into the Xho I site of x-172. The oligonucleotide sequences, rials and Methods. An M13 sequencing primer (15-mer, New their orientation, and associated P-galactosidase activities (25) are as England BioLabs) was used because this primer permits extension of follows. Oligonucleotide 1 (TGTGTATATAATA): +, 570 (R) and the cDNA from a site in lacZ located about 25 bp downstream from 1238 (D); -, 95 (R) and 266 (D). Oligonucleotide 2 (TATATAATA): the fusion point of HIS4 and lacZ. The DNA extended with this +, 696 (R) and 1293 (D); -, 233 (R) and 815 (D). Oligonucleotide 3 primer was analyzed on an 8% acrylamide sequencing gel. pBR322 (TATA): +, 39 (R) and 28 (D). Oligonucleotide 4 (TATAA): +, 119 DNA cut with Hpa II and labeled with [a-32P]dCTP and Klenow (R) and 210 (D); -, 50 (R) and 43 (D). Oligonucleotide 5 (TATAAA): fragment was used as a size marker (lane M). WT, wild type. +, 460 (R) and 980 (D); -, 48 (R) and 46 (D). Downloaded by guest on September 24, 2021 8560 Genetics: Nagawa and Fink Proc. Natl. Acad. Sci. USA 82 (1985)

TATA or TATAA fail to promote the initiation of HIS4 the oligonucleotide is inserted >100 bp upstream from the transcription, although the TATAA sequence restores a small normal TATA, either in the UAS region (lanes SA) or amount of activity. Analysis of the transcripts in the func- upstream from. the UAS (lanes 6A), there is virtually no tional oligonucleotide insertions (Fig. 3) shows that the transcription (there are, as in x-172, only very weak tran- pattern of5' ends is almost identical for each ofthe insertions scripts initiating within the HIS4 coding region). but is different from wild type. The pattern for these oligo- In the second set ofexperiments (the B series in Fig. 4) the nucleotide insertions is heterogeneous; in addition to the oligonucleotide was recombined with a HIS4 region that transcript initiating at the I site, a major transcript initiates at contains the wild-type TATA-123 to create two potentially a position 11 bp 3' from the I site and many minor ones initiate functional TATA sequences. Insertion ofthe oligonucleotide at several different positions even farther 3'. We assume that into deletion x-101 (Fig. 4, lanes 2B), located 26 bp upstream the altered transcription pattern results from the altered from TATA-123, results in multiple initiation sites-some of spacing between the oligonucleotides and the I site. In these the transcripts initiate at a variety of sites upstream from the constructions the TATA is 6-10 bp closer to the I site than normal I site and some initiate at the I site. When the it is in the wild-type sequence. This spacing and the pattern oligonucleotide is inserted 55 bp upstream from the of transcripts is similar to deletion x-131 (Figs. 2 and 3). TATA-123, the major transcript begins 51 bp upstream from The Relationship Between TATA and the I Site. The the normal I site (lanes 3B). In this construction, transcription insertion of the oligonucleotide TATATAATA at several initiates at a new upstream site despite the presence of the positions upstream from the normal TATA-123 site provides normal TATA-123 and the normal I site. Constructions in important insights into the requirements for transcription which the oligonucleotide is placed within the UAS (lanes SB) initiation (Fig. 4). In one set of experiments (the A series in or upstream from the UAS (lanes 6B) initiate like the wild Fig. 4), this oligonucleotide was inserted into a HIS4 5' type, at the normal I site, as if the upstream TATA is not region from which the normal TATA-123 site had been recognized and TATA-123 is utilized. deleted (x-172). In these constructions the TATA-123 deletion x-172 was combined with various upstream deletions DISCUSSION containing the synthetic TATA oligonucleotide. When the oligonucleotide is inserted 11 bp upstream from the site ofthe Wild-type yeast strains have a single HIS4 transcript that deleted TATA-123 (Fig. 4, lanes 2A), the HIS4 mRNA initiates 60 bp from the ATG that starts translation. Our initiates at the same site as in wild type (I). However, when analysis shows that the DNA sequence at -123 is required for the oligonucleotide is inserted either 40 bp (lanes 3A) or 55 bp initiation ofthis transcript at the I site at -60. Deletion ofthis (lanes 4A) upstream from the normal TATA-123 site, the -123 site abolishes normal transcription from I. Insertion of transcript begins 45 bp upstream from the normal I site. When the oligonucleotide TATAAA at the site of this deletion is

WT 2A 3A 4A 5A 6A 2B 3B 5B 6B - I m --er --r----I - 1nP i M R D fR D R D R D R D R D R D R D R D R D

mm -.- .- :v

Sb

6A 5A 4A 3A 2A

x-172 E I ATG- WT booA 4TATA

68 56 38 28

L ------

FIG. 4. Insertions of TATA at new sites in HIS4. The oligonucleotide TATATAATA containing Xho I cohesive ends was inserted at sites of Xho I linker insertions upstream from TATA-123 and the RNA from strains containing these insertions was analyzed as described for Fig. 2. In the diagram below the autoradiogram, the location ofeach insertion ofthis oligonucleotide is shown by a vertical arrow. These constructions were obtained by ligating two fragments: an upstream fragment containing the oligonucleotide insertion and a downstream fragment containing TATA-123. The numbers refer to the Xho I linker insertion from which the upstream fragment was obtained (see Fig. 1): 2 = x-101, 3 = x-170, 4 = x-210, 5 = x-407, and 6 = x-99. The letters refer to the downstream fragment, either A (x-172 lacks TATA-123) or B (wild type has TATA-123). The heavy horizontal black arrows show the position(s) of the major transcription starts in each construction. The open arrows are the HIS4 regulatory repeats (UAS). TATA shows the position of TATA-123. Downloaded by guest on September 24, 2021 Genetics: Nagawa and Fink Proc. Natl. Acad. Sci. USA 82 (1985) 8561

sufficient to restore initiation at the I site. This sequence direct transcription initiation requires an in vitro transcription works only in the TATAAA orientation. When we moved a system. synthetic oligonucleotide sequence upstream from the nor- mal site, the site of initiation also moved upstream (see Fig. We thank L. Guarente, E. Hoar, and S. Hahn for stimulating 4, lanes 3A and 4A), even when the normal TATA-123 was discussions and communicating unpublished data and Jef Boeke for present (Fig. 4, lanes 2B and 3B). These results show that the a critical reading of the manuscript. This work was supported by a synthetic oligonucleotide sequence contains all the informa- grant to G.R.F. from the National Institutes of Health. G.R.F. is an tion necessary for a functional TATA box. American Society Professor of Genetics. Yeast and higher cells appear to differ in the sequences that 1. Benoist, C. & Chambon, P. (1981) Nature (London) 290, control transcription initiation. In mammalian cells the tran- 304-310. script usually starts 30 bp from the TATA box, without the 2. Fromm, M. & Berg, P. (1982) J. Mol. Appl. Genet. 1, 457-481. requirement for any special sequence at the site of initiation. 3. McKnight, S. L., Gavis, E. R., Kingsbury, R. & Axel, R. In yeast the presence and position of the TATA box is (1981) 25, 358-398. necessary but not sufficient to determine the location of the 4. McKnight, S. L. & Kingsbury, R. (1982) Science 217, 316-324. transcription start site. The sequences at the start site(s) also 5. Dierks, P., Ooyen, A., Cochran, M. D., Dobkin, C., Reiser, J. contribute to the specificity of initiation. In wild-type HIS4 & Weissmann, C. (1983) Cell 32, 695-706. there is a single major transcription start at the I site. When 6. Stuart, G. W., Searle, P. F., Chen, H. Y., Brinster, R. L. & Palmiter, R. D. (1984) Proc. Natl. Acad. Sci. USA 81, the I site is deleted (Fig. 2, x-33 and x-176) or the TATA box 7318-7322. is too close or too far away from the I site (Fig. 2, x-131 and 7. Carter, A. D., Felber, B. K., Walling, M., Jubier, M. F., x-44; Fig. 4, lanes 2B), transcription starts at several different Schmidt, C. J. & Hamer, D. H. (1984) Proc. Natl. Acad. Sci. sites at various distances from the TATA, underlining the USA 81, 7392-7396. importance ofthe I site in determining the site oftranscription 8. Pelham, H. (1982) Cell 30, 517-528. initiation. However, the sequence at the I site is not neces- 9. Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. sary for transcription initiation, since deletion of this site Sci. USA 77, 1432-1436. does not abolish transcription. The sole use ofthe I site in the 10. Ghosh, P. K., Lebowitz, P., Frisque, R. J. & Gluzman. Y. presence of the other potential start sites in the HIS4 5' (1981) Proc. Natl. Acad. Sci. USA 77, 100-104. 11. Wasylyk, B., Wasylyk, C.. Augereau, P. & Chambon, P. noncoding region shows that the I site is preferred if it is (1983) Cell 32, 503-514. located at the right position relative to TATA-123. 12. Russell, P. R. (1983) Nature (London) 301, 167-169. Our data suggest that a potential site of transcription 13. Sentenac, A. & Hall, B. (1982) in The ofthe initiation will be recognized only if it is located within a Yeast Saccharomyces: and , eds. defined distance downstream from the TATA. When the Strathern, J. N., Jones, E. W. & Broach, J. R. (Cold Spring distance between TATA-123 and the I site is shortened, the Harbor Laboratory, Cold Spring Harbor, NY), pp. 561-606. 14. Burke, R. L., Tekamp-Olson, P. & Najarian, R. (1983) J. Biol. I site is not recognized and transcription begins at a new site Chem. 258, 2193-2201. about 60 bp downstream from the TATA (Fig. 2, x-44). 15. Williamson, V. M., Cox, D.. Young, E. T., Russell, D. W. & Furthermore, when the TATA is moved to new positions Smith, M. (1983) Mol. Cell. Biol. 3, 20-31. upstream from its normal location, the new site of transcrip- 16. Dobson, M. J., Tuite, M. F., Roberts, N. A., Kingsman, A. J. tion initiation is always at least 60 bp from the TATA. We also & Kingsman, S. M. (1982) Nucleic Acids Res. 10, 2625-2637. have evidence that there is a maximum distance from the 17. Holland, J. P., Labieniec, L., Swimmer, C. & Holland, M. J. TATA (1983) J. Biol. Chem. 258, 5291-5299. beyond which a potential initiation site will not be 18. Nogi, Y. & Fukasawa, T. (1983) Nucleic Acids Res. 11, recognized. When the I site is as far away as 74 bp, it is still 8555-8568. recognized (Fig. 4, lanes 2A). However, when it is 110 bp 19. Struhl, K. & Davis, R. W. (1981) J. Mol. Biol. 152, 553-568. away it is not (Fig. 4, lanes 3A and 3B). These data suggest 20. Hinnebusch, A. G. & Fink, G. R. (1983) J. Biol. Chem. 258, that the minimal spacing between the site of transcription 5238-5247. 21. Guarente. L. & Mason, T. (1983) Cell 32, 1279-1286. initiation and the TATA sequence is about 60 bp, and the 22. Donahue, T. F., Farabaugh, P. J. & Fink, G. R. (1982) Gene maximal, about 110 bp. 18, 47-59. One explanation for these distance requirements is that 23. Donahue, T. F., Daves, R. S., Lucchini, G. & Fink, G. R. RNA polymerase looks for a site about 60 bp downstream (1983) Cell 32, 89-98. from the TATA box and if it 24. Lucchini, G., Hinnebusch, A. G., Chen, C. & Fink. G. R. finds a strong start site (like the (1984) J. Mol. Cell. Biol. 4, 1326-1333. I site) it will initiate there. Ifthere is no strong start site, it will 25. Hinnebusch, A. G., Lucchini, G. & Fink, G. R. (1985) Proc. use other, weaker sites in the vicinity. This model predicts Natl. Acad. Sci. USA 82, 498-502. that in the absence of a strong start site, a yeast gene might 26. Maxam. A. M. & Gilbert, W. (1980) Methods Enzymol. 65, have many initiation sites. Perhaps those yeast genes such as 499-560. 27. Sherman, F., Fink, G. R. & Lawrence, C. W. (1974) Methods HIS] (20) and CYCI (21) that have multiple initiation sites in Yeast Genetics Laboratory Manual (Cold Spring Harbor lack a strong start site. Another explanation could be that Laboratory, Cold Spring Harbor, NY), pp. 61-64. these genes may have several functional TATA boxes (30). 28. Wolfner, M., Yep, D., Messenguy, F. & Fink, G. R. (1975) J. However, analysis of a HIS4 region with two potentially Mol. Biol. 96, 273-290. functional TATA 29. Teem. J. L. & Rosbash. M. (1983) Proc. Natl. Acad. Sci. USA sequences (Fig. 4, lanes 3B), shows that 80, 4403-4407. only the most proximal TATA is recognized. Clearly, eluci- 30. Hahn. S., Hoar, E. & Gaurente, L. (1985) Proc. Natl. Acad. dation of the mechanisms by which the TATA and the I site Sci. USA 82, 8562-8566. Downloaded by guest on September 24, 2021