Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8004-8008, December 1985 Biochemistry Footprinting of ribosomal RNA by initiation factor and RNA I (DNase I footprinting/ribosomal RNA /in vitro transcription/Acanthamoeba castellanii/eukaryotic RNA polymerase) ERIK BATEMAN, CALVIN T. IhDA, PREECHA KoWNIN, AND MARVIN R. PAULE* Department of Biochemistry and the Cellular and Molecular Biology Interdisciplinary Program, Colorado State University, Fort Collins, CO 80523 Communicated by Marshall Fixman, August 12, 1985

ABSTRACT The binding of a species-specific transcrip- preinitiation complex formed between the DNA and a tran- tion initiation factor (TIF) and purified RNA polymerase I to scription initiation factor (TIF) (6). A third region flanking the the promoter region of the 39S ribosomal RNA gene from start site is important for initiation by polymerase I (6). Since Acanthamoeba were studied by using DNase I "footprinting." polymerase is unable to initiate transcription in the absence Conditions were chosen such that the footprints obtained could of (s), it is likely that at least part of the be correlated with the transcriptional activity of the TIF- control region functions through binding of factors, with a containing fractions used and that the labeled DNA present distinct region serving as a polymerase . would itself serve as a template for transcription. The tran- DNase I "footprinting" (13) has been used as a probe for scription factor binds upstream from the transcription start a wide variety of DNA-binding , allowing identifica- site, protecting a region extending from around -14 to -67 on tion ofthose DNA sequences that are in a stable complex. We the coding strand, and -12 to -69 on the noncoding strand. have utilized this approach to determine the DNA binding The that binds to DNA within this region can be sites both for the transcription initiation factor and for competed out by using wild-type promoters but not by using purified RNA polymerase I in the transcriptional initiation mutants which do not stably bind the factor. RNA polymerase complex. Our results suggest a mechanism of promoter I can form a stable complex in the presence of DNA and recognition by polymerase in which protein-protein interac- transcription factor, allowing footprinting of the complete tion may augment protein-DNA interaction in transcription transcription initiation complex. RNA polymerase I extends the initiation. protected region obtained with TIF alone to around + 18 on the coding strand, and to +20 on the noncoding strand. This region EXPERIMENTAL is not protected by polymerase I in the absence ofTIF. The close PROCEDURES apposition of the regions protected by TIF and polymerase Preparation of TIF and RNA Polymerase I. Acanthamoeba provides evidence that accurate transcription of the ribosomal TIF was prepared by fractionation of the nuclear pellet gene may be achieved through protein-protein contacts as well obtained from the initial homogenate during preparation of as through DNA-protein interactions. RNA polymerase I (14), as described in the text. DNA Templates. Plasmids pBR322, pSBX60, pSBX60i, Transcription initiation of eukaryotic genes in vitro requires pSBX60i dl 5'-32, and pSBX60i dl 5'-26 (6) and pEBH10 were the presence of at least one protein factor in addition to RNA isolated by using standard techniques (15). Templates for polymerase and a promoter-containing DNA fragment (1-3). footprinting were end-labeled using T4 polynucleotide For class II, III, and possibly class I genes, one or more of (15), recut to give a single labeled end, and the appropriate the transcription factors acts through stable interaction with DNA fragments were purified by polyacrylamide gel elec- the gene promoter regions (4-6), allowing correct initiation trophoresis (see Fig. 1). by the polymerase. The details of this process differ consid- In Vitro Transcription. Transcription assays were per- erably between different gene classes, with respect to the formed essentially as described (6, 16), with the following sequence and positioning of promoter regions as well as the exceptions: reactions were in a final vol of 50 ILI containing number of factors thought to be required for transcription. 20mM Tris HCl, pH 7.9/150mM KCl/10 mM MgCl2/0.5 mM Control regions for polymerase II, and to some extent dithiothreitol/500 ,uM each of ATP, GTP, and UTP/5 ,uM polymerase III, show promoter sequence homology when [a-32P]CTP (10 Ci/mol; 1 Ci = 37 GBq). TIF (0-20 ,ul), 0.03 comparisons between species are made (7, 8). In contrast, the units of RNA polymerase I, 12.5 ng of pBR322/Sal I, and 2 promoter sequences involved in polymerase I transcription ng ofpurified DNA fragment were used in each assay. Assays are highly diverged, making identification of regulatory were at 27°C for 20 min. sequences by comparison of conserved regions difficult. DNase I Footprinting. Footprinting reactions were carried Studies on ribosomal gene promoters do, however, show that out under the same conditions as the transcription assays a region flanking the 5' side of the initiation start site is except and RNA polymerase were omitted, or required for transcription (9). Part ofthis region functions by they were done as indicated in the figure legends. TIF and/or interaction with protein components of the system to form a RNA polymerase I were incubated with 5000-40,000 cpm (2 stable complex that commits the template for correct tran- ng) oflabeled DNA for 15 min at 27°C prior to addition of 1-2 scription (6, 10-12). In the Acanthamoeba rRNA genes, the ,g of DNase I (Worthington DPFF) and digestion for 30 sec sequence required for template commitment extends from at 27°C. Reactions were terminated by adding NaDodSO4, around -20 to -47, and it can be divided into two regions: sodium acetate (pH 5.2), and tRNA to final concentrations of one (A region) is absolutely required for transcription, and 0.2%, 0.3 M, and 50 ug/ml, respectively. After extraction the other (B region) is involved with the stability of a with phenol and chloroform, samples were analyzed on 8% sequencing gels and by autoradiography (17). Sequencing The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: TIF, transcription initiation factor; bp, base pair(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8004 Downloaded by guest on September 30, 2021 Biochemistry: Bateman et al. Proc. Natl. Acad. Sci. USA 82 (1985) 8005 ladders produced by the G reaction of Maxam and Gilbert strate that a footprint reflects a functionally active complex (17) were used to align bands on all footprinting gels. in vitro. This may be especially important when partially purified proteins are assayed for DNA-binding under non- RESULTS standard conditions. As a control experiment, we tested the amount of tran- Templates and TIF Preparations Used. We initially attempt- scription obtained from the small amounts ofDNA fragments ed to obtain a footprint of the Acanthamoeba rDNA promot- used for the footprinting experiments described below. Fig. er by using TIF prepared by phosphocellulose chromatogra- 2 shows that accurate transcription occurs under the condi- phy of cytoplasmic (6, 16) or nuclear extracts (unpublished tions used for footprinting, demonstrating a functionally data). These preparations, while transcriptionally active, did active complex. The amount of transcription obtained re- not show protection of DNA from digestion by DNase I, flects the amount ofTIF preparation added, and it appears to presumably because of an insufficient concentration of TIF plateau late in the titer. We had previously determined that or because of nonspecific competition for DNA binding sites the amount of polymerase added was in excess, so the assay by other proteins in the extract. Accordingly, the TIF used would reflect the formation of TIF complexes. here was prepared by polyethyleneimine precipitation of a DNase I Footprinting of TIF Complexes. Workers in this 0.15 M KCl extract of the nuclear pellet from an RNA laboratory have demonstrated that at least one component, polymerase I crude extract (14), followed by chromatography other than RNA polymerase I, present in extracts is on phosphocellulose, cibacron-blue agarose, DEAE-cellu- capable of forming a stable complex with rDNA from lose, and a second phosphocellulose column. This procedure, Acanthamoeba (6). Deletion of parts of the rDNA suggested to be described in greater detail elsewhere, resulted in highly that the binding site is located within a DNA region extending active, but still impure, TIF and a purification of -20,000- from -20 to -47. If so, this region should be protected from fold relative to a whole-cell extract. DNase I digestion in the presence of TIF. A diagram of the DNA fragments used for footprinting is The pattern of bands obtained from DNase I digestion of shown in Fig. 1. All fragments were prepared from pSBX60 the promoter-containing fragment in the presence of increas- or pSBX60i, which differ only in the orientation of the insert ing amounts ofTIF is shown in Fig. 3. In the absence of TIF, (6), or pEBH10, containing a larger rDNA insert than the a continuous ladder of fragments is generated, with a few former two plasmids. Transcription ofthe first template (Fig. bands overrepresented (Fig. 3, lanes 1 and 6). In the presence 1A), proceeding toward the Nru I site of pBR322, produces of TIF, protection from digestion on the coding strand a 49-base runoff, while transcription of the second template (transcribed strand) is obtained between positions (Fig. 1B) gives a 307-base runoff, proceeding toward the Sal -14 and around -65 (Fig. 3A, lanes 2-5). The extent of I site of pBR322. The 74-base-pair (bp) insert contained in protection is dependent on the amount of TIF used in each these fragments is sufficient to support accurate transcription reaction and appears to correlate reasonably with the level of and template commitment at a level identical to templates transcription obtained with equivalent TIF concentrations containing larger rDNA inserts (16). Both pSBX60 and (Fig. 2). To maintain the overall extent of digestion constant pSBX60i were labeled at the Nru I site, allowing footprinting in the presence of various amounts of TIF, we found that it of both strands. pEBH10 was labeled at the EcoRI site for was necessary to include linearized pBR322 DNA. This had noncoding-strand footprinting or at the HindIII site for no effect on the positioning of the footprint. It is presumed coding-strand footprinting. Transcription Under Footprinting Conditions. DNase I footprinting measures the degree ofprotection from DNase I M 1 2 3 4 5 M 6 7 8 9 10 M digestion conferred by protein bound to a specific site along a uniquely end-labeled DNA fragment (13). For the results obtained to be meaningful, there must be some indication that protection of DNA is relevant to the process being studied. _--3maJ~in-307 b In many cases, DNA sequences protected are implicated in a process by genetic data, but it is still necessary to demon-

Sal I XmaIII Xrna NIW 650 -55 -47 -20 419, 973 A 49 bp-

Sal Xma 1 XnaU Nru I 660 +19 ,, -20 -47 -56 9733 B i -~~~~~ 307 bp i~~~~~~~~~~~~ EcoRl HindU -120 -47 -20 ,, 80 49b C I~~~~~~~~I Bamr I Ps1I FIG. 1. Templates used for footprinting. The drawings show the DNA fragments derived from pSBX60 (A) or pSBX60i (B) used for DNase I footprinting. Boxed areas represent the rDNA insert, and shaded areas represent the biochemically defined TIF binding FIG. 2. Transcription under footprinting conditions. Runoff tran- domain (6). The start site and direction oftranscription are indicated scription assays were carried out as described. Lanes: 1-5, tran- by an arrow. Numbers outside the boxed area are pBR322 coordi- scription of 2 ng of pSBX60 Nru I/Hinfl fragment using 0, 5, 10, 15, nates; those inside are relative to the start site. (C) EcoRI/HindlII and 20 pd ofTIF, respectively; 6-10, transcription of2 ng ofpSBX60i portion ofpEBH10. The regions outside the rDNA insert are from the Nru I/Sal I fragment using 0, 5, 10, 15, and 20 ju ofTIF, respectively. multiple-cloning site of pUC8. The rDNA was inserted at the HincHI The size and position of the runoffs are indicated. M, size markers: site of pUC8. Msp I/pSBX60i. b, Bases. Downloaded by guest on September 30, 2021 8006 Biochemistry: Bateman et al. Proc. Natl. Acad. Sci. USA 82 (1985)

A M 1 2 3 4 5 6M B M 1 2 3 4 5 6 M

-68

-51 .._ . -12

-47 -- ., ..a----_._om .0 -32 - FIG. 3. DNA protection by TIF on the rDNA -2 go --_ .... _ -14 _ 4 --,.... _~~~-4 coding and noncoding strands. (A) DNA protec- -~~~~~~~-51 tion on the coding strand was measured as de- .-47 _om _o _o 0m scribed. Open box represents the DNA region ------~~-47[ _U. . _ _ required for TIF interaction (6); arrow shows the -2 1 start site and direction of transcription, and bar -- Mm --O 6 indicates the region protected from DNase I. So. _ Numbers along the borders are the -. In -6- 8 +14 right positions of guanine residues relative to the start site. (B) _-- ---32 -2 r DNA protection by TIF on the rDNA noncoding strand. Lanes: 1 and 6, no TIF; 2-5, 5, 10, 15, and 20 Atl of TIF, respectively. M, size markers. that the effect ofcarrier DNA is to reduce nonspecific binding Binding of RNA Polymerase I to TIF Complexes. Fig. 5 of the labeled DNA by proteins present in the TIF prepara- shows the effect of adding increasing amounts of purified tion. RNA polymerase I to a TIF-DNA complex. On the coding Protection of the nontranscribed strand by TIF extends strand, polymerase extends the TIF footprint downstream to from position -12 to around -65 (Fig. 3B, lanes 2-5). position +18, which includes the transcription start site (Fig. Protection from DNase I on this strand is accompanied by 5A, lanes 3-6). The footprint was obtained at all polymerase slight enhancement of cutting at several positions between concentrations tested, even though the protection obtained is -10 and +20. In general, the protection of the noncoding not as clear as that afforded by TIF (Fig. SA, lane 2). strand is less complete than that of the coding strand, Polymerase protects a similar region on the nontranscribed although reproducible protection was obtained. We have not strand (Fig. 5B, lanes 3-6), extending the footprint obtained been able to clearly define the 5' boundary ofthe footprint on with TIF alone to position + 19. On the noncoding strand, the the nontranscribed strand because the reappearance of the bands enhanced in the presence of TIF alone are markedly control level of digestion is gradual rather than a sharp the become transition (see below). It is clear that a component ofthe TIF reduced by polymerase, and bands farther up gel preparation binds tightly to both strands of the DNA, enhanced. Addition of polymerase in the absence of TIF did protecting a region of =50 bp. DNA Binding Is Sequence Specific. We wanted to test 1 2 3 4 5 M M whether the DNA footprinting activity of the presumptive TIF had the same sequence specificity as template commit- U~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ment (6) because it is unlikely that a random interaction with DNA would show this property. In this experiment, TIF was incubated, prior to the standard footprint assay, with an excess of DNA fragments containing either the entire rDNA -65 - - _ insert or 5' deletions extending into the TIF binding region. - .x 6.0 -47 - Ifthe observed footprint results from the same DNA-protein ft--aim-_ I - 11 interaction necessary for template commitment to transcrip- .*am al -PU an _ -12 tion initiation (6), then wild-type DNA fragments should I~~~~A -20L_ Ili prevent footprinting by binding all the available TIF, while -14 I-- - -20 the deleted templates should not affect the footprint. The 5' -U ___ _-.___da_01 dl-32 deletion is able to support transcription, but it does not __14o a -m stably bind TIF. The 5' dl-26 deletion neither binds TIF nor +1T =" _"_ _W_ -_. _w_ on_ supports transcription (6). . The results of this experiment are shown in Fig. 4; lanes 4.N. -. 1 -47 1-5 are from the coding strand, and lanes 6-10 are from the _ m a _0 ".0 noncoding strand. Incubation of TIF with wild-type rDNA - "N__o .. prior to footprinting completely prevents protection from DNase I digestion (compare lanes 2 and 3 and lanes 7 and 8 in Fig. 4). Preincubation of TIF with either the 5' dl-32 or 5' dl-26 deletions did not affect the appearance of the footprint (Fig. 4, lanes 4, 5, 9, and 10). The protection of DNA by TIF must, therefore, be dependent on a small region of DNA FIG. 4. Competition for TIF footprinting activity. TIF (20 Al) was preincubated for 10 min with various prior to addition of contained within the rDNA insert; the same region we end-labeled DNA and digestion as described. Lanes: 1 and 6, no TIF; showed indirectly to function by interaction with TIF to form 2 and 7, TIF, no competitor; 3 and 8, TIF + wild type rDNA; 4 and a stable preinitiation complex (6). These results render 9, TIF + 5' dl -32 rDNA; S and 10, TIF + 5' dl -26 rDNA. M, size unlikely the possibility that the observed footprinting on both markers. Lanes 1-5 are from the coding strand and lanes 6-10 are DNA strands is due to nonspecific protein-DNA interaction. from the noncoding strand. Downloaded by guest on September 30, 2021 Biochemistry: Bateman et al. Proc. Natl. Acad. Sci. USA 82 (1985) 8007

A 6 7M B

Q. -32 FIG. 5. DNA protection by RNA polymerase I of -25 - -14 the rDNA coding and noncoding strands. (A) Footprinting reactions were performed on the coding -ma strand in the presence ofTIF and polymerase. Lanes: +1 1, no TIF; 2, TIF only; 3-6, TIF + 0.006, 0.012, 0.03, and 0.06 units of RNA polymerase I, respectively; +8 7, -_ 0.06 units of RNA polymerase I only. Bar indicates region protected by polymerase I. (B) Footprinting zs;'_ reactions were set up on the noncoding strand in the 46 ..- presence ofTIF and polymerase as follows: lane 1, no do TIF; lane 2, TIF only; lanes 3-6, TIF + 0.006, 0.012, 0.03, and 0.06 units of RNA polymerase I, respec- 65 tively; lane 7, 0.06 units of RNA polymerase I only. i__ _ ~'-3 Numbers on the right borders are the positions of I._..:.__:"'.. guanine residues relative to the start site.

not give any protection from DNase I on either strand of the insert or several base pairs into the vector sequences flanking template (Fig. 5A, lane 7; Fig. 5B, lane 7). the insert. To be sure that these borders accurately identify The Same Footprinting Pattern Is Obtained with a Wild- the ends of the rDNA sequences protected from DNase I Type rDNA Template. The borders of the footprints obtained digestion, rather than artifactual borders resulting from weak using pSBX60 and pSBX60i extend to the limits of the rDNA interaction with vector DNA, footprinting experiments were carried out on DNA fragments containing much larger rDNA A inserts. Fragments isolated from pEBH10 (Fig. 1C) contain Codina Non-coding Acanthamoeba rDNA sequences extending from -120 to 1 2 3 M M 4 5 6 +80. Fig. 6A shows the protection afforded by the TIF alone A WD% (lane 2 on the coding strand, lane 5 on the noncoding strand) and by TIF plus RNA polymerase I (lane 3 on the coding strand and lane 6 on the noncoding strand). The TIF protects TI 67 +2O-.3 .. m a similar region on this wild-type DNA as on the truncated TIF -3912e templates. The upstream borders are -67 on the coding -14 strand and -69 on the noncoding strand, and the downstream border is -14 on the coding and -12 on the noncoding strand. P0l TIF,.i, Enhanced digestion of several sites downstream of the TIF - ~8+18 footprint borders were also observed. Strong enhancements were observed at -5 and +8 on the coding strand and at -2,

_ __ ~-6a around + 10, and around + 16 on the noncoding strand, with additional weak enhancements at -6, -4, and +1 on the _ +37 noncoding strand. Addition ofRNA polymerase extended the footprint to + 18 and +20 on the coding and noncoding strands, respectively. On the noncoding strand, there is a major enhancement at approximately +35 upon addition of polymerase, while all ofthe enhancements resulting from TIF are ,mSALM 1aS. binding eliminated. DISCUSSION B We have shown that one or more components present in a -80 -60 -40 -20 +1 +20 +40 partially purified TIF preparation binds to part of the pro- moter sequence ofAcanthamoeba rDNA. The protection can be correlated with the amount of transcription supported by TIF I-4-Poi I- TIF, making it likely that a functionally active complex is present under the conditions used for footprinting. A sum- FIG. 6. DNA protection by TIF and RNA polymerase I (pol) on mary of the data is shown in Fig. 6B. The TIF footprint wild-type rDNA. (A) Footprinting reactions were performed on the extends from position -14 to -67 on the coding strand, and coding and noncoding strands of pEBH10. Lanes: -1 and 4, no from -12 to -69 on the noncoding strand. additions; 2 and 5, TIF only; 3 and 6, TIF + RNA polymerase I. Numbers indicate positions of guanine residues relative to the start The upstream portion ofthe promoter from Acanthamoeba site, shown in the marker lanes (M). (B) Summary ofDNA protection rDNA can be subdivided into two regions termed A and B (6). by TIF and RNA polymerase I. Hatched area represents TIF That these regions of the promoter affect the ability of TIF to protection; open area represents polymerase I protection. Shaded bind to DNA is directly demonstrated by results described area represents the DNA region required for template commitment here. The protected region wholly contains the TIF interac- (6). tion sequence defined biochemically (6) and extends several Downloaded by guest on September 30, 2021 8008 Biochemistry: Bateman et al. Proc. Natl. Acad. Sci. USA 82 (1985) base pairs on either side ofthis region. Competition between protein-DNA recognition events (24). One can envision the DNA templates containing either wild-type or 5'-deleted promoter recognition process to involve the formation of a promoter regions as measured by footprinting show a direct preinitiation complex between the TIF and the promoter correlation between the ability of a template to commit and DNA. This involves sequence-specific contacts between the the physical binding of a protein to the A and B promoter TIF and DNA and results in both species specificity and regions. Furthermore, the competition experiments provide template commitment. The preinitiation complex is recog- a strong indication that footprinting by the presumed TIF is nized by the polymerase through protein-protein and, per- highly sequence specific and is unlikely to result from haps, protein-DNA contacts. The various steps in the overall adventitious binding by impurities present in the TIF prep- initiation process (recognition, binding, and phosphodiester aration used. Template commitment also has been demon- bond formation) are not necessarily dependent solely on the strated in mouse and human systems (10-12). Similarly, same interactions. For example, formation of the first crude extracts of Xenopus oocytes contain DNA-binding phosphodiester bond in transcription or DNA unwinding proteins that alter digestion of rDNA promoter regions (18). could be dependent on the DNA sequence at the transcription However, the inability to demonstrate a correlation between start site while, as shown here, the initial recognition of the binding and transcriptional activity in the crude system promoter by polymerase is certainly augmented by TIF. makes interpretation of the results uncertain. What is the nature of the component responsible for This work was supported by U.S. Public Health Service Grants footprinting? The behavior of TIF on a wide variety of GM26059 and GM22580 to M.R.P. chromatography media suggest that it is a single protein; we a requirement for more than 1. Matsui, T., Segall, J., Weil, P. A. & Roeder, R. G. (1980) J. have not observed transcription Biol. Chem. 255, 11992-11996. one fraction, in addition to RNA polymerase I, following any 2. Korn, L. J. (1982) Nature (London) 295, 101-105. purification step used. These results are in contrast to those 3. Hall, B. D., Clarkson, S. G. & Tocchini-Valentini, G. (1982) obtained using mouse or human extracts, which suggest that Cell 29, 3-5. two distinct fractions support accurate rRNA transcription 4. Setzer, D. R. & Brown, D. D. (1985) J. Biol. Chem. 260, by pol 1 (12, 19, 20). The TIF giving a footprint in our system 2483-2492. appears to correspond to the species-specific DNA-binding 5. Davison, B. L., Egly, J., Mulvihill, E. R. & Chambon, P. protein described elsewhere (12, 21). The protection of -50 (1983) Nature (London) 301, 680-686. bp of DNA by TIF on both DNA strands would appear to 6. Iida, C. T., Kownin, P. & Paule, M. R. (1985) Proc. Natl. indicate that a relatively large protein or multimer is in- Acad. Sci. USA 82, 1668-1672. 7. Wickens, M. P. & Laskey, R. A. (1981) in Genetic Engineer- volved, although we acknowledge that, as in the case of ing, ed. Williamson, R. (Academic, New York), Vol. 1, pp. TFIIIa, protection ofthis size DNA fragment can be achieved 104-167. by a Mr 38,000 protein (22, 23). We have preliminary 8. Kuntzel, H., Piechulla, B. & Hahn, U. (1983) Nucleic Acids evidence, from sedimentation on glycerol gradients and size Res. 11, 893-900. exclusion chromatography, that TIF has a native Mr around 9. Sommerville, J. (1984) Nature (London) 310, 189-190. 250,000-350,000 (unpublished data). 10. Cizewski, V. & Sollner-Webb, B. (1983) Nucleic Acids Res. Purified RNA polymerase I extends the protection ob- 11, 7043-7056. tained with TIF alone by -30 bp on either strand, with no 11. Wandelt, C. & Grummt, I. (1983) Nucleic Acids Res. 11, discernible gap between the TIF footprint and that of the 3795-3809. 12. Miesfeld, R. & Arnheim, N. (1984) Mol. Cell. Biol. 4, 221-227. polymerase. The polymerase footprint extends only in the 13. Galas, D. & Schmitz, A. (1978) Nucleic Acids Res. 5, direction of the transcription unit. The footprint obtained 3157-3170. with polymerase was consistently weaker than that ofthe TIF 14. Spindler, S. R., Duester, G. L., D'Alessio, J. M. & Paule, alone; addition of more polymerase did not increase tran- M. R. (1978) J. Biol. Chem. 253, 4669-4675. scription (unpublished data) or the clarity of the footprint. 15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular One possible explanation is that the polymerase complex is Cloning: A Laboratory Manual (Cold Spring Harbor Labora- less stable than the TIF complex, allowing greater attack by tory, Cold Spring Harbor, NY). DNase I, although other structural proposals are equally 16. Paule, M. R., Iida, C. T., Perna, P. J., Harris, G. H., Brown- plausible. Since polymerase alone did not give any protection Shimer, S. & Kownin, P. (1984) Biochemistry 23, 4167-4172. of the DNA fragments, the polymerase footprint obtained in 17. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, the presence of TIF can be regarded as a transcription- 499-560. specific complex. RNA polymerase apparently is able to 18. Dunaway, M. & Reeder, R. H. (1985) Mol. Cell. Biol. 5, and bind to the TIF-DNA complex but not to 313-319. recognize 19. Cavanaugh, A. H. & Thompson, E. A., Jr. (1985) Nucleic promoter DNA alone. Acids Res. 13, 3357-3369. The juxtaposition of the TIF and polymerase footprints is 20. Cavanaugh, A. H., Gokal, P. K., Lawther, R. P. & suggestive ofclose contact between the two molecules bound Thompson, E. A., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, to the promoter. This statement must be qualified by the 718-721. possibility that the steric constraints that apply to DNase I 21. Grummt, I., Roth, E. & Paule, M. R. (1982) Nature (London) digestion of protein-DNA complexes might result in a foot- 296, 173-174. print that is larger than the actual DNA region tightly bound 22. Bieker, J. J. & Roeder, R. G. (1984) J. Biol. Chem. 259, to protein. The apparent closeness ofthe TIF and polymerase 6158-6164. I footprints nonetheless provides support for the suggestion 23. Engelke, D. R., Ng, S., Shastry, B. S. & Roeder, R. G. (1980) that promoter recognition by eukaryotic may be Cell 19, 717-728. facilitated by protein-protein interactions in addition to 24. Travers, A. (1983) Nature (London) 303, 755. Downloaded by guest on September 30, 2021