Vol. 11, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1382-1392 0270-7306/91/031382-11$02.00/0 Copyright © 1991, American Society for Microbiology

Sequences Far Downstream from the Classical tRNA Promoter Elements Bind RNA Polymerase III Transcription Factors LISA S. YOUNG,' DAVID H. RIVIER,12t AND KAREN U. SPRAGUE' 3* Institute of Molecular Biology' and Departments of Biology3 and Chemistry,2 University of Oregon, Eugene, Oregon 97403 Received 27 February 1990/Accepted 18 December 1990

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNAAIaC gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNAAIaC promoter is in contact with factors. The protected region extends from -1 to at least + 136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequencespecific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNAAlaC promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

The promoters of tRNA genes have generally been thought to consist of two small segments internal to the coding region. These promoter elements are each about 10 bp long and are called the A and B boxes (reviewed in reference 18). This picture was originally derived from studies of the transcriptional properties of mutant tRNA genes that had undergone partial deletion (8, 16, 34). It has been supported by the effects of some point mutations within the A and B boxes (reviewed in reference 18). The idea of a two-element tRNA promoter gained additional appeal from early reports that two distinct transcription factor fractions were required, along with polymerase III, for tRNA transcription. A straightforward synthesis of these data led to a model for tRNA transcription in which two transcription factors, called TFIIIB and TFIIIC, activate tRNA genes by binding to the A and B boxes, respectively (11, 28, 35; see also reference 25). In contrast to this view, work from our laboratory shows that the full tRNA promoter extends far beyond the A and B boxes and that the machinery that acts upon it includes more than two transcription factors. In particular, DNA sequences completely outside the coding region of a Bombyx silkworm tRNAA'IaC gene are required for full transcriptional activity in vitro (40). The promoter for this gene occupies a region of -160 contiguous bp, including the entire coding region plus at least 13 bp upstream of the transcription start site and at least 48 bp downstream of the transcription termination site. Moreover, we have shown that in the case of the silkworm, the transcription apparatus consists of at least four components, each of which is absolutely required (29). These components are polymerase III and transcription factors TFIIIB, TFIIIC, and TFIIID. Several pieces of evidence suggest that this degree of complexity is not limited to the tRNA gene that we have Corresponding author. t Present address: Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720. *

examined. First, the transcriptional properties of tRNA genes in a variety of other organisms (including yeast, Drosophila and Xenopus species, and humans) are influenced by sequences outside the A and B boxes (reviewed in reference 18). Second, there is evidence for additional components in the polymerase III transcription machinery in other organisms. Specifically, the TFIIIC-containing fraction from human cells has been subdivided into two fractions (called TFIIIC1 and TFIIIC2), both of which are required for transcription (10, 41). Furthermore, yeast TFIIIC fractions contain multiple polypeptides that comigrate as a complex with tRNA genes (4, 14, 30). At least three of these polypeptides are required for transcriptional activity (30), and at least four can be cross-linked to the template (4). Although the relationship of the silkworm transcription components to the fractions described in other systems is not completely understood, an economical interpretation that takes chromatographic similarities into account is that the silkworm fractions designated polymerase III and TFIIIB correspond to the fractions with those names in other systems, whereas the silkworm fractions called TFIIIC and TFIIID are included in the fractions that are elsewhere called TFIIIC or Tau (18, 29). Whether silkworm TFIIIC and TFIIID correspond to human TFIIIC1 and TFIIIC2 is not known. Viewed as a whole, the current data suggest that the simple model of two transcription factors bound to two small sites within the coding region may be inadequate to describe tRNA gene transcription. Additional transcription factors and more extensive DNA sequences seem to be involved. To understand the function of all of the elements that contribute to the extended tRNA promoter, we have begun a systematic examination of the interaction between the transcription components that we have resolved and a silkworm tRNAAlaC gene. Previous work from our laboratory showed that the full tRNAAlaC control region consists of at least two functionally distinct domains (Fig. 1). The downstream coding/3'-flanking domain (from about +5 to + 146) includes both of the classical tRNA promoter elements (the A and B boxes) in 1382

tRNA GENE 3'-FLANKING SEQUENCES BIND TFIIIC/D

VOL. 11, 1991

I

a

I

by DNase I footprinting that the combined factors TFIIIC and TFIIID interact with a portion of the tRNAAlaC control region that closely matches the coding/3'-flanking domain. Moreover, mutational alteration of segments of this large domain demonstrates that both halves of it are essential for binding these factors.

T

A

B

-

+98

+1

1383

FULL TRANSCRIPTIONAL ACTIVITY

FULL COMPETITION STRENGTH

b -30

-40

-20

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TAATAAGACTTTATATrAGTAA1TlllGCAAGCTITllTTTCT +40

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+70

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9TATarrA1TCGAAACGArCATMATTrMGCAATCATrrCTTTATAAATCACATrrCGATT +160

+170

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+190

+200

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CAATTCAA1TTGGACMATAATATTMATACTATTATTCTMACGAACTACCAACCTCG

FIG. 1. (a) Diagram of the tRNAAlaC gene. The rectangle represents the primary transcript from the gene. Filled areas denote the A and B boxes, and the brackets below indicate the extent of sequences required either for full transcriptional activity or for full competitive strength. I and T represent the sites of initiation and termination of the primary transcript. (b) Nucleotide sequence of the noncoding strand of the gene, numbered from the site of transcription initiation (19). Within the sequence, the boxed area delimits the primary transcript, the A box is underlined, and the normal B box and three flanking, degenerate B boxes are underlined in boldface.

addition to sequences downstream of the transcription termination site. The upstream domain (from at least -13 to about +5) consists mostly of sequences 5' to the transcription initiation site but may also include a short stretch (-5 bp) between the initiation site and the A box (40). Although both domains are required for transcriptional activity of the gene, they are distinguishable on the basis of their contribution to competitive strength in crude extracts. The coding/ 3'-flanking domain affects the ability of the gene to compete for one or more of the transcription factors that is limiting in crude extracts. In contrast, the upstream region either does not affect competition for transcription factors at all or does so for factors that are not limiting in typical crude extracts (40). In this report, we examine the interaction of the silkworm transcription factors TFIIIC and TFIIID with the tRNAAlaC gene to determine whether either of the previously identified functional domains corresponds to the TFIIIC/D binding site. We have focused on this subset of factors because template exclusion experiments had earlier identified it as one that is capable of binding stably to the tRNAAlaC gene (29). In contrast, binding by resolved TFIIIC and TFIIID fractions was not detectable in those experiments. The TFIIIB/D subset also binds tRNAAlaC genes stably (29), but the detailed analysis of this interaction is complicated by our recent discovery of a novel functional component in the TFIIIB fraction (43). Since the TFIIIC/D-containing fractions are free of this novel component, analysis of the TFIIIC/D-gene interaction is, at present, more straightforward than is analysis of the TFIIIB/D interaction. We show

MATERIALS AND METHODS Preparation of fractions containing TFIIIC and TFIIID. Silkgland nuclear extract (40) (40 ml; 4 mg of protein per ml) was loaded onto a 60-ml DEAE-Sephadex (Sigma A-25-120) column 2.5 cm in diameter equilibrated in buffer B (50 mM Tris-HCl [pH 7.5], 0.2 mM EDTA, 1 mM dithiothreitol, 20% glycerol) plus 250 mM KCl and washed with the same buffer. Protein-containing fractions in the flowthrough with an A2./ A280 ratio of 0.7x peak height) eluting at 750 mM KCl were pooled, dialyzed against buffer A (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 20% glycerol, except where noted) plus 180 mM KCl and called C+D/2 column. Since large amounts of this fraction could be prepared easily, it was used for most experiments. To produce smaller amounts of cleaner fractions containing both TFIIIC and TFIIID, we used a modification of our earlier procedure designed to separate TFIIIC from TFIIID (29). By deliberately overloading the Mono S column relative to the amounts of total protein that allow separation of TFIIIC and TFIIID, we could obtain a highly active TFIIIC/ D-containing fraction that retained less than 1% of the protein in the 750 mM KCl phosphocellulose step fraction. A 4.5-ml sample of the dialyzed 750 mM KCl phosphocellulose step fraction (5 mg of protein) was loaded onto an HR 5/5 Mono S column and eluted with a gradient of KCl as described previously (29). Fractions were dialyzed against buffer A plus 125 mM KCl and 10% glycerol and then assayed for TFIIIC and TFIIID activity. A series of fractions from 375 to 400 mM KCl were pooled and called C+D/3 column. This pool typically retains 80% of the TFIIID activity and 50% of the TFIIIC activity present in the preceding phosphocellulose fraction and contains approximately 200-fold less protein. To obtain separated TFIIIC and TFIIID, 0.9 ml (1 mg) of the dialyzed phosphocellulose 750 mM KCl fraction was loaded onto an HR 5/5 Mono S column and eluted with a gradient of salt as described previously (29). Assays for transcription components. Individual components were detected by complementation with appropriate subsets of the transcription machinery as described elsewhere (29). The complementing fractions are typically of intermediate purity and in some cases have low levels of the activity being tested. The TFIIIC and TFIIID activities in all fractions used for binding experiments were quantitated in single-round assays (21) under conditions in which polymerase III and the other required factors were in excess, and the transcription activity was linearly dependent on the added factor fraction. To limit transcription to one round, heparin was added to transcription complexes stalled at position +8 by omission of CTP. Full-length transcripts were produced when CTP was provided along with heparin. We used RNA fingerprinting techniques (3) to verify that the correct stalled

1384

YOUNG ET AL.

oligonucleotide and primary transcript (19) were synthesized. The molar yields of products from reactions with and without CTP demonstrated that the stalled oligonucleotide was quantitatively converted to full-length transcript in the presence of heparin. Therefore, the assay provides a reliable measure of active transcription complexes formed on the wild-type tRNAAIaC gene. Cloned genes. The genes used to generate fragments for DNA binding experiments and DNase I footprinting have been described previously (23). These were a wild-type tRNAAIaC gene inserted in a smaller version of pBR322 (pWT ALA 3') and its derivatives deleted from the 3' end, as well as a wild-type tRNAAlaC gene inserted in standard pBR322 (pWT ALA 5') and its derivatives deleted sequentially from the 5' end. The wild-type DNA sequence from -65 to + 121 of the tRNAAlaC gene has been reported previously (36). The additional sequence from + 122 to +218 shown in Fig. 1 was determined for both strands, using partial chemical cleavage (27) and synthesis in the presence of chain-terminating nucleotides (32). Preparation of labeled fragments. Fragments from the 5' deletion series were generated by digestion with AvaI and AatII (to yield a 336-bp fragment from the wild-type gene, a 293-bp fragment from 5'A -3, a 263-bp fragment from 5'A +27, and a 200-bp fragment from 5'A +90). Fragments from the 3' deletion series were generated by digestion with AccI and AatII (to yield a 487-bp fragment from the wild-type gene, a 396-bp fragment from 3'A +124, a 361-bp fragment from 3'A +89, and a 316-bp fragment from 3'A +44). Labeling was at the AvaI site for the 5' deletions and at the AccI site for the 3' deletions. For footprinting, the 437-bp AvaI-PvuII fragment from pWT ALA 5' was labeled on either strand at the AvaI site. Labeling at the 5' end was with [-y-32P]ATP and polynucleotide kinase (27); labeling at the 3' end was with [X_-32P]dCTP and the Klenow fragment of Escherichia coli DNA polymerase I (Boehringer Mannheim) (26). After labeling and isolation, the concentration of each fragment preparation was determined by ethidium bromide staining of samples run with standards on a 4% polyacrylamide gel. Electrophoretic DNA binding assay. Binding reactions contained between 5 and 40 fmol of labeled fragment, either 4,000 ng of unlabeled poly(dI-dC) (when C+ D/2 column was used) or 200 ng of poly(dI-dC) (when C+D/3 column was used), and 7.5 ,ul of the TFIIIC/D fraction in a total volume of 20 ,u. Details of the reactions with separated TFIIIC and TFIIID are given in the figure legends. The final concentrations of buffer components were 70 mM KCl, 30 mM Tris-HCl (pH 7.5), 4 mM MgCl2, 10% glycerol, and 3 mM dithiothreitol. To achieve binding, it was essential to include at least 1 mM dithiothreitol in the binding reactions. Dependence on reduced thiols has also been reported for binding of human TFIIIC (9). Reactions were initiated by addition of the TFIIIC/D fraction and incubated at room temperature for 30 min. The entire reaction mixture was loaded onto a 3.5% polyacrylamide gel (1:30 bisacrylamide/acrylamide) in a buffer containing 0.05 M Tris-borate (pH 8.0) and 5 mM EDTA, and the products were electrophoresed at 150 V for 3 h at room temperature (until marker bromphenol blue run separately had reached 12 cm). Gels were dried and exposed to Kodak XAR-5 film overnight (-16 h) with an intensifying screen. Amounts of fragment in the bound and unbound positions were quantitated by scintillation counting of excised gel slices in Ecolume scintillant (ICN Biomedicals). Binding efficiency was measured directly by calculating the fraction of total labeled fragment that migrated in the bound

MOL. CELL. BIOL.

position. Binding efficiency was measured competitively by using the linear form of the competition curve (35) to compute the competitive strengths of mutant genes relative to the wild-type gene at each competitor/labeled fragment

ratio. The interaction measured in these experiments appears to be equilibrium binding on the basis of two criteria. First, a time course showed that complex formation on wild-type genes was complete within the incubation period used. The amount of complex increased during the first 10 min of incubation but did not change from 10 min to 120 min of further incubation. Second, the formation of complexes was reversible. Addition of a molar excess of unlabeled template to preformed complexes disrupted them completely within 20 min. DNase I footprinting. When gene-factor complexes were to be footprinted, the 30-min binding reactions were followed by a 2-min treatment with 25 ng of DNase I (Worthington) in standard binding reaction conditions (see above). DNase I digestion was stopped with EDTA at a final concentration of 5 mM, and the products were electrophoresed as described above. Bound fragments were detected by exposing the wet gel overnight at room temperature to Kodak XAR-5 film. Gene-factor complexes were eluted from gel slices as described previously (15) except that the elution buffer contained 0.1% sodium dodecyl sulfate. Eluted fragments were phenol extracted, ethanol precipitated, and electrophoresed on standard 5% polyacrylamide sequencing gels (1:30 bisacrylamide/acrylamide) containing 8 M urea, until the marker xylene cyanol FF dye reached 16 to 20 cm. Gels were washed in 5% acetic acid-5% methanol to remove urea, dried, and exposed for 2 to 30 days at -70°C with an

intensifying screen.

RESULTS Binding of factors TFIIIC and TFIIID alters the mobility of fragments bearing the tRNAAIaC gene. In previous analyses of the interaction between tRNAAIaC genes and transcription factors TFIIIC and TFIIID, binding was measured indirectly in template exclusion assays (29). These experiments showed that in the silkworm transcription system, combinations of at least two factors are required for detectable binding to the gene. Such factor combinations must include TFIIID, and either binary combination that does so (TFIIIC plus TFIIID or TFIIIB plus TFIIID) is stable and capable of initiating the formation of an active transcription

complex. To investigate binding more directly, we have exploited the fact that protein binding frequently alters the electrophoretic mobility of small DNA fragments (12, 17). Fragments bearing tRNAAIaC genes were incubated with fractions

that contained both factors TFIIIC and TFIIID but lacked TFIIIB and polymerase III. Figure 2 shows assays of these activities in the fractions used. The electrophoretic mobility of gene-containing fragments was reduced by the same amount when either of two fractions provided the transcription factors (Fig. 3A). One of these fractions (C+D/3 column) was purified by gradient elution from Mono S. It was enriched for TFIIIC and TFIIID over total protein by at least 100-fold compared with the other fraction, a phosphocellulose salt step (C+D/2 column). Thus, it is likely that complex formation with TFIIIC and TFIIID was responsible for the observed reduction in fragment mobility. The pattern of three closely spaced bands is reproducible, although the relative proportions of the bands vary in different factor

tRNA GENE 3'-FLANKING SEQUENCES BIND TFIIIC/D

VOL. 11, 1991 A

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FIG. 2. Evidence that the phosphocellulose 750 mM salt step (C+D/2 column) and pooled Mono S (C+D/3 column) fractions contain TFIIIC and TFIIID activity but no TFIIIB or RNA polymerase III activity. Shown are the results of multiple-round complementation assays for individual transcription factors. (A) TFIIIC assay; (B) TFIIID assay; (C) TFIIIB assay; (D) RNA polymerase III assay. Transcription products were analyzed on 8% polyacrylamide gels. Each assay included a negative control in which all components were present except the one being tested (-), a previously calibrated source of the appropriate component as a positive control (+), and the two test fractions. 0, Origin; T, position of tRNAAIaC transcripts.

preparations. The presence of each of the bands was dependent on tRNA gene sequence, since each was efficiently subject to competition by plasmid DNA bearing wild-type tRNAAIaC genes but not by plasmid sequences alone (Fig. 3B). We used a quantitative approach to test more rigorously the idea that TFIIIC and TFIIID are responsible for the tRNAAJaC gene-protein complexes. Since neither of these components is homogeneous, complex formation could, in principle, be due to unrelated proteins. First, we determined whether the amount of bound complex observed with a particular phosphocellulose fraction corresponded to the amount of TFIIIC or TFIIID activity actually present in the fraction. We quantitated the activity of each transcription factor in assays that limit transcription to a single round (see Materials and Methods). Under conditions in which all components but the one being tested are provided in excess,

F

FIG. 3. Electrophoretic DNA binding assay with fractions containing TFIIIC/D. (A) Comparison of the protein-bound fragments observed with TFIIIC/D-containing fractions from the phosphocellulose 750 mM KCI salt step (C+D/2 column) with those derived from the Mono S column (C+D/3 column). Each reaction mixture contained 10 fmol of 32P-labeled fragment. (B) C+D/2 column-pWT ALA 5' gene complexes (20 fmol of 32P-labeled fragment in each reaction mixture) that are detectable in the presence (+) of increasing amounts of either gene-containing or vector plasmid DNA as a competitor. Lanes marked - correspond to reaction mixtures that contained neither of these competitors. All reaction mixtures contained sufficient poly(dI-dC) to eliminate fragment retardation due to nonspecific DNA-binding proteins (see Materials and Methods) and to maintain a constant concentration of total nucleic acid. The mole ratio of competitor to 32P-labeled fragment was 1, 2, or 7.5. At these competitor-fragment ratios, gene-containing competitor reduced the number of bound complexes to 58, 28, and 12.5%, respectively, of the value observed with no competitor. At these same ratios, vector competitor reduced the number of complexes to 89, 95, and 79% of the value observed with no competitor. 0, Origin; F, position of free DNA fragment. Arrows indicate the three electrophoretically distinct species of bound fragments.

the number of transcripts obtained after one round of transcription measures the number of active transcription complexes that can be formed per unit test factor. The TFIIIC and TFIIID activities of three preparations of a phosphocellulose 750 mM KCI fraction were measured and were compared with the amounts of gene-protein complex detected electrophoretically with each preparation. The DNA binding activity of these fractions varied but in each case was very close to the TFIIID activity (Table 1). Since TFIIID plays a pivotal role in binding (29), and since all of the phosphocellulose fractions tested were TFIIID limited, it is reasonable that the amount of bound complex should be determined by the amount of TFIIID activity. Second, we used highly purified preparations of separated

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YOUNG ET AL.

1386

TABLE 1. Relationship between DNA binding activity and transcription factor activity in fractions containing TFIIIC and TFIIID' Transcription factor activity (fmol of active transcription complex/4dl of fraction)

DNA binding activity (fmol of gene-protein complex/pLl of fraction)

Fraction

TFIIID

TFIIIC

0.025 0.073 0.087

0.14 0.13 0.22

.4.

0.033 0.073 0.070

1 2 3

a Three different preparations of the material that elutes from phosphocellulose at 750 mM KCI (see Materials and Methods) were tested. DNA binding was measured by quantitating gene-protein complexes that had been electrophoretically resolved from free DNA; transcription factor activity was measured by quantitating active transcription complexes in single-round transcription assays limited for each test factor (see Materials and Methods).

TFIIIC and TFIIID to perform reconstruction experiments. That is, resolved TFIIIC and TFIIID, whose activities had been quantitated in single-round transcription assays, were mixed in amounts calculated to provide TFIIIC and TFIIID activities equal to those in a particular phosphocellulose fraction. The ability of the reconstituted TFIIIC/D mixture to form complexes on tRNAAlaC genes was compared with that of the unresolved factors in the phosphocellulose fraction. Identical amounts of bound complex were generated either by mixing separated factors or by adding a single, cruder fraction that contained both activities (Fig. 4). We examined the three phosphocellulose 750 mM KCl preparations described in Table 1 and two different preparations of

FragIm

Comparison of gene-factor complexes obtained with a mixture of separated TFIIIC and TFIIID. Shown are the bound fragments generated either by a phosphocellulose 750 mM KCI fraction containing both TFIIIC and TFIIID (C+ D) or by separated TFIIIC and TFIIID [C, D (separatFIG. 4.

unfractionated TFIIIC/D and with

ed)] that

were

quantitations

equivalent to the amounts phosphocellulose fraction. Factor single-round transcription assays as

combined in amounts to be

of each factor present were

in

based

the on

described in Materials and Methods.

Each reaction mixture

con-

activity and 0.12 fmol of TFIIID activity 1.29 fmol of TFIIIC activity and 0.36 fmol of TFIIID activity

tained 0.43 fmol of TFIIIC

(A)

or

32 P-labeled DNA (B). All reaction mixtures contained 10 fMol Of fragment and 4,000 ng of poly(dl-dC). Positions of the origin (0), free DNA fragments (F), and bound fragments are indicated.

3Bound *.ragments

mm. : __W

FIG. 5. Evidence that TFIIIC and TFIIID are both required to detect gene-factor complexes. Shown are results of an electrophoretic DNA binding assay with 0.2 fmol of a separated TFIIID fraction (D alone), 0.5 fmol of a separated TFIIIC fraction (C alone), or a mixture of 0.5 fmol of TFIIIC and 0.2 fmol of TFIIID (C+D). Each reaction mixture contained 20 fmol of 32P-labeled DNA fragment and 600 ng of poly(dI-dC). Positions of the origin (0), free DNA fragments (F), and gene-specific bound fragments are shown. The band marked with an arrow is discussed in the text and represents a nonspecific complex that forms with the TFIIIC fraction alone as well as with the combination of TFIIIC plus TFIIID.

TFIIIC and TFIIID in this manner (not shown). In all cases, the reconstituted TFIIIC/D mixture had the same complexforming ability as the phosphocellulose fraction. As a final test of the connection between TFIIIC/D and the complexes on tRNAAIaC genes, we determined whether complex formation was dependent on the presence of both factors. Previous template exclusion experiments had established that neither TFIIIC nor TFIIID alone is sufficient for detectable binding to the gene (29). The presence of both factors is required. Figure 5 shows the results of incubating TFIIIC and TFIIID with gene-containing DNA fragments, either separately or in combination. Neither fraction alone generates the standard TFIIIC/D-gene complex. The significance of the rapidly migrating complex that is formed with the TFIIIC fraction is not known. If it is due to TFIIIC binding, the interaction is extremely nonspecific. It is efficiently subject to competition by both vector and gene sequences (data not shown). The key result is that formation of the gene-specific complex requires both TFIIIC and TFIIID. Control experiments (not shown) established that the slowly migrating complexes are identical in mobility to those formed with unresolved TFIIIC and TFIIID and are subject to competition by gene sequences but not by vector sequences. Combined factors C and D protect a large region of the tRNAAIaC gene from DNase I cleavage. To learn where the TFIIIC/D factor complex is bound on the tRNAAlaC gene, we determined which sequences are protected from DNase I cleavage in the presence of fractions containing TFIIIC and TFIIID. Since we do not have conditions in which all of the gene-containing fragments are bound in solution, we separated bound from unbound fragments after treating with DNase but before examining the DNase I cleavage pattern (see Materials and Methods). Sequences extending over a large region (approximately -1 to at least +136) on both strands are protected (Fig. 6A). The footprint has a bipartite appearance; a region between positions + 80 and + 107 on the

tRNA GENE 3'-FLANKING SEQUENCES BIND TFIIIC/D

VOL . 11l 1991

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FIG. 6. DNase I footprint of TFIIIC/D fractions on the wild-type tRNAAlaC gene. (A) Footprints of the C+D/2 column fraction on the coding or noncoding strand of the 437-bp pWT ALA 5' fragment. The extent of sequences required for full transcriptional activity (see Fig. 1) is indicated by a vertical rectangle within which filled areas represent the A and B boxes. The outer two lanes show the products of DNase I cleavage of free DNA. The inner two lanes show the products of DNase I cleavage of bound fragments. Well-protected segments on each strand are bracketed. The positions of initiation (I) and termination (T) of the primary transcript are marked. (B) Footprint of the C+D/3 column fraction on the noncoding strand of the same fragment. Symbols are as in panel A.

coding strand and positions +88 and +111 on the noncoding strand shows less complete protection. In addition, certain positions on the noncoding strand (-1 and +75) are hypersensitive to DNase I cleavage. To test the possibility that the unusually large size of this footprint was due to adventitious binding by proteins unrelated to factors TFIIIC and TFIIID, we compared the DNase I protection patterns produced by the phosphocellulose (C+D/2 column) and Mono S (C+D/3 column) fractions described in the preceding section. Both fractions give identical DNase I footprints on both strands. A representative footprint formed by the Mono S fraction on the noncoding DNA strand is shown in Fig. 6B. With both protein fractions, the two parts of the footprint are clearly visible, and their relative intensities are the same. Thus, it is likely that the entire footprint is the consequence of interaction with TFIIIC/D. We have also compared the DNase I footprints of TFIIIC/ D-gene complexes that differ in electrophoretic mobility by eluting each band separately after DNase treatment and electrophoresis. The complexes corresponding to the three bands in Fig. 3 were analyzed separately and found to have DNase I protection patterns that were indistinguishable from

each other and from the footprint of the mixture of complexes shown in Fig. 3 (data not shown). Although the relative proportions of these complexes vary with different preparations of transcription factors, we have not found a correlation between the presence of one type of complex and the level of TFIIIC or TFIIID activity measured in independent transcription assays (data not shown). Such a correlation has been reported for the complexes formed with human TFIIIC (20). Sequences in both parts of the DNase I footprint contribute to TFIIC/D binding. To determine whether DNA sequences throughout the region protected from DNase I cleavage are actually required for TFIIIC/D binding, we tested the effect of sequence alterations in this region. Using electrophoretic DNA binding assays, the affinities of wild-type and mutant genes for factors TFIIIC/D were compared in two ways. First, we measured direct binding to labeled DNA fragments; second, we measured the ability of unlabeled DNA sequences to compete for TFIIICID binding to labeled DNA fragments. To measure direct binding, we prepared labeled DNA fragments that contained either wild-type or partially deleted tRNAAlaC genes and quantitated the ability of these frag-

1388

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TABLE 2. Quantitation of TFIIIC/D binding to wild-type and mutant tRNAAIaC genes' % of wild-type binding

Deletion

endpoint

Ni

Wild type 3'A+ 124

3'A+89 3'A+44 5'A-3 5'A+27 5'A+90

a

Measured

Measured

directlyb 100 34 (+5) 10 (+1) 10 (+3) 95 (±3) 54 (±2) 10 (±3)

competitivelyc 100 38 (33-44) 6 (5-7) 2 (0.2-4) 78 (75-81) 56 (49-62) Undetectable (

-13 A

iI B

I.I

n

+146 0

B

in

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m

F

FIG. 8. Competition of DNA binding by partially deleted tRNAAIaC genes. Each reaction mixture contained 20 fmol of labeled fragment and either no competitor (0) or 40 fmol of either the vector plasmid without a tRNAAlaC gene (V) or plasmids bearing wild-type or partially deleted tRNAAlaC genes. Vertical rectangles above each lane represent the wild-type (WT) and mutant promoters of these genes, as described in the legend to Fig. 7. 0, Origin; B, position of bound fragments; F, position of free DNA fragments.

containing DNA (Fig. 8 and Table 2). Figure 8A shows the effect of deleting sequences from the 3' end of the tRNAA'IaC gene. Deletion to position + 124 reduces competitive strength to 38% of the wild-type value, and deletion to position +89 or position +44 reduces it still further, to levels (6 and 2%, respectively, of the wild-type value) that are barely distinguishable from competition by vector sequences (lane V in Fig. 8). Thus, as was the case for measurements of direct binding to mutant genes (Fig. 7), removal of sequences corresponding to the 3'-flanking segment of the TFIIIC/D footprint drastically reduces the ability of tRNAAlaC genes to compete for TFIIIC/D binding. Likewise, removal of sequences from the 5' end of the gene affects competitive strength in the same way that it affects direct binding of TFIIIC/D. Removal of 5'-flanking sequences to -3 has little effect on competitive strength, whereas removal of the A box (5'A +27) reduces competitive strength to 56% of the wild-type value, and removal of additional sequences that include both the A and B boxes (5'A +90) reduces competitive strength to that of vector sequences alone (Fig. 8B). DISCUSSION We have investigated the interaction between a silkworm tRNAAIaC gene and two components of the silkworm transcription machinery, TFIIIC and TFIIID. When these two factors are combined, a complex is formed with the gene that can be detected by retardation of the electrophoretic mobility of gene-bearing DNA fragments. Neither TFIIIC nor TFIIID alone produces this complex. Using DNase I footprinting, we have determined which sequences within the

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large promoter of this gene are bound in the presence of TFIIIC and TFIIID. A motivation for this analysis was the possibility that stable contacts with transcription factors are made only through the A and B boxes and that sequences outside these elements play a kinetic role, perhaps by increasing the likelihood of the initial interaction between template molecules and transcription factors. Such a role has been suggested for sequences upstream of a Drosophila tRNAArg gene (33). An alternative possibility is that sequences throughout the entire promoter make direct and stable contact with transcription factors. Our data reveal an extensive region devoted to stable factor binding and fit closely with the predictions from our previous functional analyses of the tRNAAlaC gene. The basic idea that had emerged from those experiments, namely, that the promoter of this gene is large and bifunctional, is supported by the features of the TFIIIC/D binding site described here. First, the large size of the region devoted to binding factors TFIIIC and TFIIID provides a simple explanation for most of the extent of the tRNAAIaC promoter. Second, the location of the TFIIIC/D binding region gives substance to the idea that the tRNAAlaC promoter consists of at least two functional domains. The TFIIIC/D binding region does not occupy the upstream domain of the promoter, but corresponds closely to the previously identified coding/3'-flanking domain (40). Our experiments reveal direct contacts between TFIIIC/D and the entire coding/3'flanking domain and thus eliminate the necessity for invoking a special and purely kinetic role for the 3'-flanking portion of this domain. Indeed, direct analysis has not revealed any kinetic effects of sequences in the coding/3'flanking domain. Examination of mutant genes at time intervals that were either longer or shorter than the time required for equilibrium binding to the wild-type gene did not reveal differences in the abilities of the mutant genes to form complexes (42). This result suggests that the mutant phenotype is simply the consequence of reduced affinity for TFIIIC/D. We do not yet know whether reduced affinity results from loss of contacts with polypeptides in both the TFIIIC and TFIIID fractions or only one of them. The entity that is in direct contact with DNA might be present in only one of these fractions but might require a component in the other fraction in order to bind DNA. Whether or not polypeptides in both fractions contact the wild-type template directly, it is interesting to consider how complexes formed on mutant and wild-type templates differ. Mutations in the TFIIIC/D binding region clearly reduce the number of TFIIIC gene complexes, but do they also alter these complexes qualitatively? The similar electrophoretic mobilities of complexes formed on wild-type and mutant genes are consistent with the possibility that these two classes of complexes contain the same proteins. This interpretation is supported by our observation that transcription of the 3' deletion mutant +89 requires all of the components that are essential for transcription of wild-type genes (38a). Thus, qualitative differences between the complexes formed on mutant and wild-type genes may be quite subtle. Specific protein-DNA contacts are presumably lost at the sites of sequence alteration in the mutant templates, yet proteins that normally bind wild-type versions of such sites may remain part of the transcription complex. The association of such proteins with mutant templates could be mediated by contacts with other polypeptides whose template binding sites are still intact. Our results thus suggest that multiple transcription factors interact with each other to function as a unit when binding to

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the downstream tRNAAlaC promoter domain. Interaction with a multifactor complex could explain why transcription from genes mutant in the downstream promoter domain can be driven to wild-type levels at high concentrations of template (39, 40). If the same multifactor complexes bind to both wild-type and mutant templates, but simply bind with reduced affinity to the mutant genes, then increasing the concentration of mutant templates should eventually produce the same number of functional complexes as are formed on wild-type templates at a lower concentration. Thus, at concentrations of template that are high enough to be saturating for wild-type genes, mutant and wild-type templates could form the same number of qualitatively similar transcription complexes and thus direct transcription at identical rates (37). Protein-protein interactions among transcription factors also provide a basis for understanding the quantitative difference between the effects of mutation on TFIIIC/D binding and transcriptional activity. Clearly, the effect on TFIIIC/D binding is larger than the effect on transcriptional activity. For instance, the 3' deletion mutation at +89 reduces TFIIIC/D binding by at least 10-fold, whereas it reduces transcription by only 2-fold (40). A plausible explanation for this discrepancy is that TFIIIC/D binding is measured in the presence of the two factors alone, whereas transcriptional activity is measured in the presence of the complete transcription apparatus. It is likely that interactions with other transcription components can partially compensate for the loss of specific contacts between TFIIIC/D and the template. This interpretation is supported by the finding in other laboratories that TFIIIB stabilizes the binding of TFIIIC to certain templates (24; reviewed in reference 18). In contrast to sequences in the coding/3'-flanking domain, those in the upstream domain, particularly those upstream of the transcription start site, must play some role other than direct interaction with TFIIIC/D. Deletion of these sequences has a dramatic effect on transcriptional activity but essentially no effect on equilibrium TFIIIC/D binding. Preliminary kinetic analysis suggests that upstream sequences do not affect the rate of TFIIIC/D binding (42). Thus, we favor the idea that the upstream domain has a distinct function, possibly to provide sequence-specific contacts for TFIIIB/D or polymerase III. A sequence nonspecific role in TFIIIB binding has recently been proposed for DNA in this part of a yeast tRNA gene (21). It is remarkable that in the silkworm, the TFIIIC/D binding region includes both coding sequences encompassing the A and B boxes and noncoding sequences that are significantly downstream of these canonical class III promoter elements. The size of the silkworm TFIIIC/D binding region clearly exceeds the prediction of the simple A and B box model. In this light, we note that there are other examples in which protection by the TFIIIC fraction extends beyond the B box. In a yeast tRNA gene, for example, sequences surrounding both the B box and the A box are protected from DNase I digestion by a TFIIIC fraction (38). A comparable fraction from human (HeLa) cells protects nearly all of the coding region of a silkworm tRNAAIaC gene (from -1 to +74) (13). In the case where a TFIIIC fraction from human (293) cells has been resolved into two distinct activities, one of these by itself (TFIIIC2) protects a region somewhat larger than the classical B box (10, 41), and addition of the other activity (TFIIIC1) extends the protection to include the A region. The unusual feature of the silkworm TFIIIC/D binding site

MOL. CELL. BIOL.

is that it includes sequences far outside the coding region. One way to rationalize the disparity between our factor binding results and those in other systems is to suppose that the preparations of TFIIIC from other organisms correspond to only one of the factors that we have examined (TFIIIC or TFIIID) and thus bind a smaller region than is bound by the combined factors. This explanation is not entirely satisfactory, since the stable binding generally exhibited by such TFIIIC fractions in template exclusion assays (2, 7, 24, 31) suggests that they contribute activities equivalent to those of both silkworm TFIIIC and TFIIID rather than to that of only one of these factors. A second possibility is that even in other systems, the regions that contribute to TFIIIC binding are actually more extensive than was first appreciated. As indicated above, published footprints already establish that the TFIIIC binding site is not restricted to the B box. Upon close examination of these data, there are hints that TFIIIC protection may extend downstream as well (see reference 7, for example). The fact that few published footprints permit thorough scrutiny of regions far outside the A/B box region is not surprising. In the absence of prior data demonstrating their functional importance, the contribution of 3'-flanking sequences to transcription factor footprints would have been attributed to binding by extraneous proteins and therefore ignored. The functional importance of some promoter elements could easily have been missed, however, since the conditions commonly used for in vitro transcription can make even the canonical promoter elements appear unnecessary (39, 40). In fact, there is already evidence that 3'-flanking sequences are likely to bind transcription factors in at least two other class III systems: (i) sequences downstream of the termination site in a Drosophila tRNAArg gene have been shown to affect stable association with unfractionated polymerase III transcription machinery (33), and (ii) 3'-flanking sequences are essential for yeast U6 promoter function both in vivo and in vitro (6). It is clear from the data presented in this report that sequences distinct from the A and B boxes contribute directly to transcription factor binding in tRNA genes. Thus, it is unlikely that transcription of tRNA genes is controlled entirely by two small sites within the coding region. Although the simplicity of this idea was appealing, the inclusion of flanking sequences in the factor binding region yields a model that may actually be more biologically reasonable. One of the shortcomings of the two-site/two-factor model in its simplest form was its inability to account for large differences in the transcription rates of different natural tRNA genes (see reference 22, for example). The extreme sequence conservation of the A and B boxes (16) suggests that sequence polymorphism in these elements alone is unlikely to provide enough variation in transcription factor affinity to account for differences in transcriptional activity. Moreover, although the spacing between the A and B boxes does vary, this parameter appears to have little effect on transcription factor binding or transcriptional activity (1, 11). In contrast, if TFIIIC/D binding is influenced by sequences that are free of the constraints of tRNA coding regions, it is reasonable to suppose that natural variation in such sequences could produce genes with a wide range of affinities for these factors. Thus, flanking sequences could provide the intrinsic heterogeneity necessary to achieve differential transcription of individual tRNA genes. Flanking promoter elements could consist of variant A/B box motifs or unrelated sequences. In this light, it will be interesting to determine

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whether the degenerate B boxes (see Fig. 1) in the tRNAAIaC downstream promoter domain have functional significance. ACKNOWLEDGMENTS

We are grateful to Jerry Johnson and members of his laboratory and to Rick Baker for advice and material during our initial efforts to detect transcription factor complexes on tRNA genes. We also thank Peter Kinsey and Yvonne Hall for silkworm rearing and all of the members of our laboratory for enlightening discussions and critical reading of the manuscript. This work was supported by National Institutes of Health research grants GM25388 to K.U.S. and GM32851 to K.U.S. and L.S.Y. and by a Genetics Training Grant predoctoral fellowship (GM07413) to D.H.R. REFERENCES

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