Structure and function of the S1 nuclease-sensitive site in the adenovirus late promoter

Cell, Vol. 45, 743-751,

June 6, 1966, Copyright

0 1966 by Cell Press

Structure and Function of the Sl Nuclease-Sensitive Site in the Adenovirus Late Promoter Yie-Teh Yu’ and James L. Manley Department of Biological Sciences Columbia University New York, New York 10027

Summary We analyzed an Sl nuclease-sensitive site present in supercoiled, but not linear, recombinant plasmids containing the adenovirus late promoter. Sl nicking was detected on both strands, primarily in the TATA box. Analysis of deletion mutants showed that sequences upstream of -47 and downstream of -12 are not required for Sl cutting. H.owever, a number of different base substitution mutations in stretches of G residues upstream and/or downstream of the TATA box were sufficient to eliminate Sl cutting. When the transcriptional activities of these mutant promoters were assayed in viva, six of seven mutants lacking the ability to form the Sl-sensitive structure showed no reduction in transcriptional potentlal. In fact, several showed increased promoter activities. These data show that the Sl nuclease cutting site in the adenovirus late promoter has precise nucleotide sequence requirements for Its formation. However, the ability of recombinant plasmids to adapt this conformation in vitro is not necessary for such plasmids to serve as templates for transcription in vlvo. Introduction The control of gene expression is mediated to a large degree by regulation of transcription initiation. While it is well established that in both prokaryotes and eukaryotes the sequences of bases in normal B-DNA provide important recognition signals for RNA polymerases and/or transcription factors, evidence has accumulated in recent years suggesting that non-B-DNA structures may also constitute important features of transcriptional control regions. Indeed, fifteen years ago Crick (1971) proposed that controlling regions in the chromosomes of higher eukaryotes consist largely of unpaired stretches of DNA. Since then, particularly in the last several years, evidence has accumulated suggesting the existence of a variety of different possible non-B-DNA structures in regions immediately 5’ to mRNA coding sequences (Larsen and Weintraub, 1982; Nickel and Felsenfeld, 1983; Goding and Russell, 1983; Mace et al., 1983; Nordheim and Rich, 1983; Shon et al., 1983; Evans et al., 1984). Sl nuclease has been a useful tool in detecting many non-B-DNA structures, such as cruciforms (Lilley, 1980) and junctions between B- and Z-DNA (Singleton et al., 1982) as well as other structures (see Hentschel, 1982). * Present address: Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115.

The adenovirus late promoter contains an Sl nuclease-sensitive site in nuclei isolated from either transformed cells containing the late promoter (Larsen and Weintraub, 1982) or infected cells (Weintraub, 1985) and in supercoiled recombinant plasmids (Larsen and Weintraub, 1982). In both instances, the Sl cutting site has been mapped very close to the TATA box (Goding and Russell, 1983; Weintraub, 1985). The adenovirus late promoter has been well studied in both in vivo and in vitro transcriptional assays. Two elements appear to be of primary importance. One is the TATA box, located -25 to -31 bp from the transcription start site (Corden et al., 1980; Hu and Manley, 1981; Concino et al., 1984). An additional element is located farther upstream, between -51 and -88 (Hen et al., 1982; Jove and Manley, 1984; Yu and Manley, 1984; Miyamoto et al., 1984). In vivo, the promoter appears to require for activity the presence of either an enhancer element in cis, such as the SV40 72 bp repeated sequence (Hen et al., 1982; Lewis and Manley, 1985) or a trans-activating protein, such as an adenovirus ElA gene product. Sequences in addition to the two elements described above, which encompass the cap site of the late promoter, are required for EIA-mediated activation (Lewis and Manley, 1985). The Sl nuclease-sensitive site in the adenovirus late promoter is, within the limits of resolution of the methods used, coincident with one of the essential elements of this promoter. This fact led us to design experiments, using a collection of mutants harboring base substitutions and deletions, to gain further insight into the nature and possible function of this site. In this work, we describe some of the requirements for generating the site, which support a model involving unidirectional slippage of the DNA strands past each other and base pairing between complementary copies of a direct repeat, and discuss experiments that suggest a possible function for this structure in controlling gene expression. Results To analyze in detail certain properties of the adenovirus late promoter, we first constructed the plasmid pPlOSVAbee: shown in Figure la. Fused to the late promoter are SV40 DNA sequences that encode a variant SV40 T antigen as a marker gene (see Experimental Procedures). T antigen is capable of activating heterologous promoters (e.g., Alwine, 1985); however, we do not believe that this fact compromises its use as an assayable gene, for reasons discussed previously (Lewis and Manley, 1985) and in Experimental Procedures. This plasmid and derivatives were used to study nuclease Sl sensitivity in vitro and promoter activity in vivo. Characterization of the Nuclease Sl-Sensitive Site in the Adenovirus Late Promoter Previous studies have demonstrated that supercoiled recombinant plasmids containing segments of adenovirus

Cdl 744

Ad 2 Late. Promoter

a e

SV40Ait

igen

Pst Pst p2lOSVAbee’

SV40

EAhancer

b

~2.70 81.54

SO.75 0.50

Figure 1. Structure and Sl Sensitivity of p2lOSVAbee’ (a) The adenovirus 2 major late promoter (from -36 to +33) was linked to the SV40 T antigen coding sequence. Two copies of the SV40 enhancer (Lewis and Manley, 1985) were inserted at the Sall site of pER322. RI indicates an EcoRl site; Barn, BarnHI; Fsl, Pstl. (b) The Sl sensitivity of p21OSVAbee’in either supercoiled or linear form was analyzed. Lane 1, supercoiled DNA was treated with Sl nuclease (see Experimental Procedures) and then digested with Pstl; lane 2, supercoiled DNA was linearized with Pstl prior to treatment with Sl; lane 3, PM-linearized DNA. DNA fragments were separated on an 0.9% agarose gel containing ethidium bromide. Arrows indicate the fragments resulting from Sl cleavage. M denotes DNA size markers.

DNA including tha late promoter can be cleaved by Sl nuclease at a site (or sites) near the TATA box (Larsen and Weintraub, 1982; Goding and Russell, 1983). To determine whether p21OSVAbee’ contains such a site, super-

coiled DNA was treated with Sl nuclease (see Experimental Procedures) and digested with Pstl. The products were resolved by agarose gel electrophoresis (Figure lb, lane 1). Two bands (indicated by arrows) not detected when the plasmid was digested with Pstl alone (Figure lb, lane 3) or with Pstl prior to Sl nuclease (lane 2) are apparent. The sizes of these fragments place a strong Sl cutting site in the vicinity of the late promoter. These results make several points. First, sequences between -88 and +33 are sufficient to generate the Sl cutting site (see below). Second, as with most Sl cutting sites (but not all: see Hentschel, 1982; Htun et al., 1984) the S&sensitive site in the late promoter requires superhelical tension. No cutting was detected when the DNA had been linearized prior to incubation with Si nuclease. Third, we note that Slsensitive sites have been reported to exist in the SV40 72 bp repeats (Evans et al., 1984). Had p21OSVAbee’ been cleaved in the enhancer sequence by Sl, subbands of approximately 1000 and 3000 bp would have been detected following Pstl digestion. Such bands cannot be observed in Figure lb, nor have we detected them in other experiments. This result is indicative of the hierarchical nature of many Sl nuclease-sensitive sites that are dependent on DNA supercoiling, and it suggests that the site in the adenovirus late promoter is considerably more susceptible to Sl cutting than is the site in the SV40 enhancer. Fine Mapping of Sl Nicking Sites Although previous studies had mapped the Sl cutting site in the late promoter near the TATA box, we wished to map it more precisely, to determine the number of cleavage sites, and to ask whether one or both strands are initially nicked. To address these questions, the plasmid pXB210, which is similar to p2lOSVAbee’ but contains no SV40 DNA sequences (see Hu and Manley, 1981), was treated with Sl nuclease for a short time, so that no linear molecules were produced and the majority of the molecules were nicked. This DNA was then digested with Hindlll and treated either with the large fragment of DNA polymerase to label the noncoding strand or with T4 polynucleotide kinase to label the coding strand (see Experimental Procedures). The DNA fragments were resolved on a sequencing polyacrylamide gel alongside DNA size markers obtained from a separate dideoxy sequencing experiment. The results (Figure 2a) show a collection of labeled fragments that are present on both strands, but not in the Sl-lacking controls. Calculation of the sizes of the Sl-generated fragments allowed us to define the sites at which Sl nuclease nicked each strand of the DNA (indicated in the schematic shown in Figure 2b). We note that on both strands the major nicking sites appear to be within the TATA box, although cutting was detected somewhat upstream of the TATA box on the noncoding strand and somewhat downstream of it on the coding strand. Effects of Mutations on Formation of the Sl Nuclease-Sensitive Site To gain insight into the nature of the nucleotide sequences required to generate Sl sensitivity in the adenovirus late promoter, we examined the potentials of a number of

Ad 2 Sl Nuclease-Sensitive Sites 745

a G

Figure 2. Fine Mapping of Sl Nicking Sites in the Late Promoter (a) Plasmid pxB210 was treated with Sl nuclease, linearized with Hindlll, and 5” or 3’- endlabeled as described in Experimental Procedures. The ssP-labeled samples were denatured and analyzed on a 8% DNA sequencing gel along with DNA sequencing ladders as single-stranded DNA size markers. M denotes 5’-end-labeled fragments from a pBR322 Hinfl digest; lane 1, no Sl control of lane 2; lane 2: S-end-labeled late promoter fragments (coding strand); lane 3, no Sl control of lane 4; lane 4, r-end-labeled late promoter fragments (noncoding strand). G and A are from DNA sequencing reactions of mp9-210 using dideoxy sequencing procedures (Yu and Manley, 1984). (b) A schematic indicating S&sensitive sites on the coding and noncoding strands of the late promoter, as observed in (a). Vertical lines indicate the Sl cleavage sites. The length of each line reflects the intensity of the corresponding band on the autoradiogram.

A

221\ 22v

b

5' 3'

5’ deletion

3’

deletion

I -mm

M

-29

-12

-2

+7

+5

I +7 -4.24 - 278 - 2.39 - 1.54 - 1.23

- 0.75 -0.50

recombinant plasmids containing mutations within the promoter region to be cleaved by Sl nuclease (Figure 3 and Figure 4). For these experiments, several different recombinant plasmids, or in some cases Ml3 RF DNA, were used. The various DNAs are described in the figure legends and in Experimental Procedures. In all backgrounds tested, the S&sensitive site in the late promoter was the dominant point of cleavage.

Figure 3. Sl Sensitivity of the Late Promoter: Deletion Mutants DNA samples were treated with Sl, digested with Pstl, and analyzed on an 0.9% agarose gel containing ethidium bromide as described in Experimental Procedures. The arrows indicate the DNA fragments resulting from Sl cleavage in the late promoter, and the dot indicates a fragment resulting from Sl cleavage in pBR322 sequences (see text). The following plasmids containing deleted late promoters were tested: Lane 1, pxB881; lane 2, pxB808; lane 3, pxB215; lane 4, pxB212; lane 5, pHB310; lane 6, pHB222; lane 7, p307SVAe; lane 8, p308SVAe; lane 9, p208SVA; lane 10, p308SVA. DNA samples analyzed in lanes l-8 are from Hu and Manley (1981); those in lanes 7-10 are from Lewis and Manley (1985). M denotes DNA size markers. The end points of each deletion is indicated at the top. Indicated sizes are in kilobases.

The Sl sensitivities of plasmids containing deletions of late promoter sequences were examined first (Figure 3). Supercoiled plasmids were treated with Sl nuclease and digested with Pstl. The products were resolved by agarose gel electrophoresis. Deletion from the 5’ side of the promoter to position -47 did not affect formation of the Slsensitive site, indicating that the upstream element of the promoter (Jove and Manley, 1984; Miyamoto et al., 1984)

Cdl 746

424

Figure 4. Sl Sensitivity of the Late Promoter: Base Substitution Mutants Plasmid p2tOSVAbee’and corresponding base substitution mutants (see Table 1) were treated with Si, digested by Pstl, and analyzed on an OS% agarose gel containing ethidium bromide. The plasmids tested are shown on the top of each lane. Arrows indicate the DNA fragments resulting from Sl cleavage in the late promoter, and the dot indicates a fragment resulting from cleavage in pBR322 sequences. M denotes DNA markers.

-

2.78 2.38 l 1.54 1.23 -

Table 1. In Vitro Sl Sensitivity and Relative In Vivo Transcriptional Efficiencies of the Late Promoter and Base Substitution Mutants

1 la 25 28

30 31 34 35 37 46 55 57 6263

+

A A

AAA

A A

A

A

A AAA

A

A

AAA

A

A

A

AA AA

8.90

A A AA AA

A

AA

AA

A

co.1

A

A

A A

1.13 1.17

A A

AAA

A

A A

A

A

A A AA

+

A

AA A

NO

0.45 1.72 3.57

A

A

A

A

ND

-

ND

+

do.08

+

0.24

+

0.87

The nucleotide sequence of the late promoter from -70 to + 10 is shown at the top of the table. The G to A transitions in each mutant are also listed (Vu and Manley, 1984). The promoter activities of the late promoter and mutants in 293 cells were assayed as described in Figure 5. Autoradiograms were scanned to determine the relative transcriptional efficiencies, and the transcriptional efficiency of p21OSVAbee’promoter was taken to be 1. The relative transcriptional efficiencies of these plasmids were also determined in HeLa cells, and the results were similar. ND: not determined.

does not play a role in establishing the Sl sensitivity of the late promoter. A further deletion that removes the TATA box (to -23) did, however, eliminate Sl sensitivity. Deletions extending from the 3’side of the promoter to as far as position -12 did not affect Sl sensitivity. Again, a further deletion that removed the TATA box prevented Sl cutting in the late promoter. In Figure 3 and Figure 4, a less intense subband was detected in many of the samples (indicated by a dot). The size of this fragment suggests that it arises by cleavage of pBR322 at a previously identified cruciform structure that is sensitive to Sl cutting in supercoiled plasmids (Lilley, 1980). lb examine further the requirements for formation of the

Sl-sensitive site in the late promoter, we assayed for its presence in a number of different mutant plasmids containing base substitutions (G to A transitions on the noncoding strand). Figure 4 displays the results of an experiment in which a series of mutants derived from p21OSVAbee’were analyzed for Sl sensitivity. The adenovirus sequences in these plasmids had been transferred from the original Ml3 vectors c/u and Manley, 1984) to facilitate analysis of their transcriptional potential in vivo (see below). Surprisingly, all the mutants with changes in the G strings either immediately upstream or downstream of the TATA box no longer displayed Sl sensitivity in the late promoter. The base changes of all the mutants are in-

Ad 2 Sl Nuclease-Sensitive Sites 747

123

4

5

6

78

9M

NT ,122

76

67

34 Figure 5. Promoter Activities of the Late Promoter and Mutants in 293 Cells Total cytoplasmic RNA was isolated from 293 cells transfected with p210SVAbee’or corresponding base substitution mutants (Table 1). Ten micrograms of RNA from each sample was assayed for the specific RNA transcript from the late promoter by primer-extension, and the products were analyzed on an 6% DNA sequencing gel. The arrow indicates the S-end-labeled extended product corresponding to initiation from the late promoter. The DNAs analyzed were, from lane 1 to 9: p21OSVAbee’;mutant 18; 35; 26; 34; 55; 57; 6263; and pBR322. M denotes DNA size markers.

dicated in Table 1. These findings prompted us to test three additional mutants (1, 37, and 46) with base changes in the G strings, and the results (not shown) are indicated in Table 1. These experiments lead to the conclusion that formation of the Sl nuclease-sensitive site in the adenovirus late promoter requires some, but not all, of the G residues flanking the TATA box. All the G residues necessary for generating the S&sensitive site are contained in one or the other copy of the direct repeat 5’-AAGGGGG-3: which is present from base pairs -20 to -26 and -34 to -40. Analysis of the Promoter Activity of Base Substltutlon Mutants To determine the abilities of the base substitution-containing mutants analyzed above for Sl sensitivity (see Figure 4) to initiate transcription from the late promoter in

vivo, we tested a number of them in transient expression assays. For the experiments described here, the piasmids that had been derived from p2lOSVAbee’ by directly replacing the wild-type adenovirus with mutant promoter sequences were used. Plasmids were assayed for expression in both HeLa and 293 cells. The latter are human embryonic kidney cells that were transformed by and constitutiveiy express the ElA and ElB genes of adenovirus 5 (Graham et al., 1977; Aiello et al., 1979). We chose to examine expression in both of these cells lines because our previous studies indicate that the sequences required for transcription initiation from the late promoter are different in these two ceil lines, apparently reflecting the requirement for an enhancer in HeLa ceils and an ElA gene product in 293 cells (Lewis and Manley, 1985). We therefore wished to determine whether any of the base substitutions affected promoter activity and whether any differential effects could be observed. Total cytoplasmic RNA was extracted from transfected ceils and used as a template for reverse transcription, with the primer being a single-stranded DNA corresponding to SV40 nucleotides 5094 to 5131. cDNAs were purified and analyzed by electrophoresis in 8% polyacryiamide sequencing gels. The results of a typical experiment are shown in Figure 5. All mutants, in both cell types, were tested on multiple occasions to control for any possible variations in transfection efficiency, etc. Relative transcription efficiencies are summarized in Table 1. Several points emerge from this analysis. First, the mutants behaved identically in HeLa and 293 cells, suggesting that none of the mutated G residues play a role in the differential promoter requirements we observed previously. This is consistent with the fact that the important sequences for this response, detected previously by analysis of deletion mutants, appear to be located near the mRNA start site, a region essentially devoid of guanosines on the noncoding strand (see Table 1). Second, four of the ten mutants tested led to a decrease in the amount of specific transcript produced. Two of these, single base changes at -55 and -57, had been identified previously as down mutations in in vitro transcription assays (Vu and Manley, 1984) and they appear to alter important residues of the upstream element of the late promoter. A third mutant, 31, also showed reduced promoter activity because of a change in the upstream promoter element. The only other mutant with reduced transcriptional potential was number 18, which contains three consecutive G to A transitions immediately downstream of the TATA box, thereby creating a stretch of seven consecutive adenosine residues. Third, three of the mutants, 25,28, and 30, showed 2- to 8-fold increased levels of transcription. These mutants ail have multiple transitions in the G cluster immediately upstream of the TATA box. Finally, of the seven mutants that destroy the ability of the supercoiled piasmids to display the Sl nuciease-sensitive site in vitro (see Figure 4) only one (18) showed any diminution in transcrip tion. This finding suggests that the ability of recombinant plasmids containing the late promoter to adapt an Slsensitive conformation in vitro is not required for the same plasmids to be transcriptionally active in vivo.

Cdl 746

-20

-4,0

c

-3P

CCTG~AGGGGGGCkaT*GGGG;TGGGGG

GGACTTCCCCCCGbI_AJLI;TCCCCCACCCCC

LA T --A

‘A+Ai

T

GGGC G T -G A --=G T’. -‘A A’-

A G” G

A%% .-----.

or c C C cccc

A T A

CTGGGGGGGTGG GACkTCCCC&
Figure 6. Slipped Structure of the Adenovirus Late Promoter The nucleotide sequence of the region of the late promoter surrounding the Sl nicking sites is shown at the top. Numbers indicate the position of bases relative to the mRNA start site, +l. The TATAbox is enclosed in dashed lines, and the direct repeats are indicated by arrows. The two possible “slipped” structures are drawn below. The regions of base pairing between complementary strands of the direct repeats are boxed. The small arrows indicate the sites of Si nicking, the locations of which support preferential formation of the structure on the right.

Discussion Evidence has accumulated during the last several years that nominally double-stranded duplex DNA molecules can adapt, under near physiological conditions, structures different from B-DNA. The first such structures identified were cruciforms resulting from base pairing between .closely spaced, inverted repeats in supercoiled DNA (Lilley, 1980; Panayotatos and Wells, 1981). However, additional non-B structures can also form in duplex DNA. For example, at least two sequences exist that can be cleaved by Sl nuclease in linear DNA. One of these is located upstream of a sea urchin histone gene (Hentschel, 1982) and the other, downstream of a human Ul gene (Htun et al., 1984). Both cleavage sites consist of the sequence (dC-dT),(dA-dG),. The authors suggested that they result from either denaturation and slippage of the strands past each other followed by reformation of out-of-register base pairs (Hentschel, 1982) or denaturation stabilized by extensive base stacking on the purine-rich strand (Htun et al., 1984). Recently, however, evidence was presented (Pulleyblank et al., 1985) that, in supercoiled plasmids containing a synthetic (dC-dT),(dA-dG), sequence, this sequence can form an unusual duplex structure in which protonated dC resjdues base pair with dG residues by a type of non-Watson-Crick’pairing originally proposed by Hoogsteen (1983). A number of different proposals have been put forth to explain other Sl nuclease-sensitive

sites in supercoiled DNA molecules. These include lefthanded DNA of an undefined sort, proposed to explain Sl sites formed at homopurine-homopyrimidine stretches (Cantor and Efstratiadis, 1984), slipped base pairs involving closely spaced, direct repeats upstream of Drosophila heat shock genes (Mace et al., 1983), and the borders between B- and Z-DNA (Singleton et al., 1982). The functions that DNA sequences capable of displaying Sl sensitivity in vitro might serve in vivo is not clear; however, two lines of evidence have suggested that some of these sequences might play a role in controlling gene expression. First, the location of such sites map to the 5’flanking sequences of a number of genes (Larsen and Weintraub, 1982; Goding and Russell, 1983; Mace et al., 1983; Schon et al., 1983; Htun et al., 1984; Ruiz-Carillo, 1984; Evans et al., 1984). Second, when the question has been addressed, the same sites that are Sl-sensitive in plasmids are also Sl-sensitive in chromatin when nuclei are treated with the nuclease (Larsen and Weintraub, 1982; Nickel and Felsenfeld, 1983; Weintraub, 1983; Weintraub, 1985). The data presented here suggest a specific structure for the Sl nuclease site in the adenovirus late promoter and a model for how it might function to regulate expression. The adenovirus late promoter contains a significant inverted repeat sequence including residues from -26 to -12 and from -7 to +8 (Ziff and Evans, 1978). The potential of this sequence to form a stem-loop structure was suggested by Goding and Russell (1983) to be responsible for the Sl cutting they and Larsen and Weintraub (1982) had detected. However, other explanations have also been put forth. Mace et al., (1983) noted the existence of direct repeats flanking the TATA box and suggested that strand slippage might be responsible for the Sl sensitivity. Ruiz-Carillo (1984) proposed that the (dC-dT).(dA-dG) sequence near the mRNA start point could create an Sl cutting site. Our mutational analyses, however, appear to rule out the involvement of both the inverted repeat sequence and the (dC-dT)(dA-dG) stretch. Additionally, since all of the mutations studied were base transitions, the unequal distribution of purine and pyrimidine residues between the two strands was not altered. Therefore, the Slsensitive structure is not simply the result of a homopurine-homopyrimidine stretch in the DNA (Evans et al., 1984; Cantor and Efstratiadis, 1984). We believe that our data support the notion that the Slsensitive structure in the late promoter results from strand slippage involving the direct repeats flanking the TATA box. Specifically, we suggest that the 5’-AAGGGGG-3’sequence can slip in torsionally constrained molecules and that Sl sensitivity is due to the creation of two singlestranded bubbles resulting from out-of-register base pairing (Figure 6). This model is supported by the studies with deletion mutants and by the observation that only base substitutions changing the G residues that form part of the direct repeats eliminated Sl cutting. The model is further strengthened by the location of the predominant Sl nicking sites (Figure 6). These sites cluster primarily in the two single-strand loops that would result from strand slippage. Interestingly, although slippage could in principle occur in either of two directions, the Sl nicking data are most con-

$:92 Sl Nuclease-Sensitive Sites

s&tent with slippage in one direction only. Although this model is strongly supported by our data, confirmation will require analysis of additional point mutations in this region. The effects of the point mutations on transcription from the late promoter in vivo were determined in transient expression assays. The results are, for the most part, consistent with our previous analysis of these mutants in an in vitro transcription system c/u and Manley, 1964). Single base changes at -55 or -57 were significantly down (by factors of approximately 10 and 4, respectively)-a result similar to, though greater in magnitude than, what we had observed in vitro. Likewise, a double mutation at positions -62 and -63 had no detectable effect in vitro or in vivo. This result is significant because this mutation alters the final two bases of an inverted dyad repeat sequence located between position -52 and -63. Based on certain properties of late promoter deletion mutants in vitro, we had proposed that an element of the late promoter separate from the TATA box is situated between -51 and -66 (Jove and Manley, 1964). Recently, this idea has been confirmed by Sawadogo and Roeder (1965) and Carthew et al. (1965) who identified and characterized a transcription factor that binds to this region of DNA. Thus, our results suggest that in vivo and in vitro, the entire palindrome is not required for transcription, and therefore, presumably, is not required for binding of this factor. Of the six mutants that destroy the Sl nuclease sensitivity in the late promoter and do not affect any other known promoter element, and that were tested for transcription in vivo, only one (mutant 16) showed a reduced level of RNA synthesis, and this was only a small reduction (see Table 1). However, three mutants showed reproducibly elevated levels of transcription. From these results, we conclude that the potential of late promoter-containing supercoiled plasmids to display an Sl nuclease-sensitive site in vitro is not a prerequisite for the promoter to be active in vivo. Indeed, the fact that several of the mutants that are no longer Sl-sensitive showed elevated levels of transcription, coupled with the structure of the Sl-sensitive site that we propose, suggests a model in which formation of the Sl sensitive structure could function to inhibit transcription. In the “slipped” structure shown in Figure 6, the TATA box region of the DNA is not only denatured, but the two strands are physically removed from each other in separate loops. We suggest that in this conformation, the TATA box binding factor, identified by Davison et al. (1963) is unable to bind to the adenovirus late promoter TATA box. This factor is required for transcription initiation to occur from this as well as other promoters, and it appears to function by binding to the TATA box, apparently as S-DNA, and forming part of a stable initiation complex. Disruption of the TATA box by slippage might thereby provide a simple mechanism for turning down the activity of the strong late promoter. Thus, mutations that prevent the late promoter from flipping to the Sl-sensitive form, but which leave sequences important for promoter function unchanged, might be expected to be up mutations. The properties of mutants 25, 26, and 30 are consistent with this prediction.

The type of regulation we have proposed may play a role in controlling expression from the late promoter during the course of a productive adenovirus infection. This promoter is known to be used inefficiently during the early phase of infection (e.g., Shaw and Ziff, 1961) but is extremely strong following DNA replication. During the early phase, the adenovirus chromatin structure could be such that the late promoter is in a slipped, Sl-sensitive configuration, perhaps as a result of protein-DNA interactions that maintain this region of the chromosome in a constrained, supercoiled conformation. The activity of the promoter would therefore be low. During DNA replication, the protein-DNA interactions that maintain this constrained configuration would be disrupted, resulting in the formation of S-DNA in this region. The late promoter would then be available for binding of the TATA box factor and for formation of stable transcription complexes, resulting in high levels of expression from the late promoter. Experimental Procedures Restriction enzymes, Sl nuclease, and avian reverse transcriptase were purchased from New England Biolab (Beverly, MA), Boehringer Mannheim (Indianapolis, IN), and Life Sciences Inc. (St. Petersburg, FL), respectively. Constructlon of Plasmids Plasmid p208b (obtained from Shu-Lok Hu), which contains a deleted W40 T antigen coding sequence linked to a mutant adenovirus late promoter in pBR322, was used as a starting plasmid. Twenty-five base pairs of coding sequence was deleted from the .SV40A gene, which results in initiation of translation at the next (in-frame) AUG codon, leading to synthesis of a T antigen lacking 13 NHrterminal amino acids. Plasmid p208bee’ was generated from p208b by inserting two copies of the SV40 72 bp repeated sequence (Lewis and Manley, 1985) at the pBR322 Sall site. The adenovitus DNA in p208bee’was removed and replaced with a wild-type (-88 to +33) or a mutated c/u and Manley, 1984) late promoter to form p21OSVAbee’and the corresponding mutants (see Figure la). The nucleotide sequence at the junction linking the late promoter and the SV40 A gene was confirmed by MaxamGilbert DNA sequencing (1980). In Vltm Sl Ssnsltlvlty Analyses DNA samples assayed for Si sensitivity were at least 80% form I. The standard reaction mixture contained 10 ug of DNA in 100 III of Sl buffer (30 mM NaOAc, pH 4.5; 300 mM NaCI; 0.2 mM EDTA; and 3 mM ZnCls; and the Sl treatment was carried out at 5 U of Sl per ug of DNA for 25 min at 42°C (Larsen and Weintraub, 19s2). After this Sl treatment, approximately half of the DNA was converted to linear (form Ill), and the other half, to nicked circles (form II). Sl was removed by phenol/CHCIs extraction. Following ethanol precipitation, DNAs were digested with the appropriate restriction enzyme and analyzed by agarose gel electrophoresis to locate the Sl cleavage site. Fine Mapping of Sl Nicking Sltas In the WlMQpa Late Promoter The sites of Sl nicking in the late promoter were mapped according to the procedures of Schon et al. (1983). Plasmid pXB210, which contains late promoter sequences from -88 to +I93 (Hu and Manley, 1981)was used in these studies. To introduce Si nicks, 12.5 ug of DNA (90% form I) in 250 ul of Sl buffer was treated with 33 U of Sl for 45 set at 3pc. After Si treatment, approximately 70% of the DNA was converted to form II and the rest remained as form I. Control DNA was treated in the same manner except that the Sl was omitted from the reaction mixture. Following phenof/CHCIs extraction and ethanol precipitation, the DNA was linearized with Hindlll (cleavage site at +193 of the late promoter), and 5’phosphates were removed with calf intestinal phosphatase. The coding and noncoding strands of late promoter were 32P-labeled at the Hindlll site by treatment with DNA polymerase I, large fragment, and T4 polynucleotide kinase, respectively (Maniatis et al., 1982). The Sl-sensitive sites in both strands were then

Cdl 750

identified by analyzing the 32P-labeled DNA samples on an 8% polyacrylamide sequencing gel (Maxam and Gilbert, 1980) with DNA sequencing ladders as size markers. ‘Ranslant Expmaakm Aaaaya Plasmid DNA was transfected into either 293 or Hela cells (on 150 mm plates) by the DNA-calcium phosphate coprecipitation procedure. exactly as described (Lewis and Manley, 1985). Total cytoplasmic RNA was isolated 48 hr after transfection, and IO pg of RNA from each transfection experiment was analyzed for the specific RNA transcript from the late promoter by primer-extension (&Knight and Kingsbury, 1982). using a 37 nucleotide B’and-labeled synthetic primer, extending from SV4O nucleotide 5094 to 5131 (Buchman et al., 1981). Extended products were resolved on 8% sequencing gals and visualized by overnight exposure to X-ray film. Autoradiograms were quanthated by densitometry scanning. To control for variations, experiments were repeated on multiple occasions with different plasmid preparations. T antigen is able to increase expression from certain promoters under some conditions (e.g., Alwine, 1985; Yu et al., unpublished data); however, this fact does not compromise its use as a test gene in the experiments described here. Thus, several of the mutant promoters were attached to the bacterial chloramphanicol acetyttransferase (CAT) gene and assayed for CAT activity following transfection in Hela or 293 cells. All mutants displayed the same relative promoter activities by this assay as described above. Acknowladgmenta We thank W. Ehrman for technical assistance, D. Read for help in constructing plasmids, E. D. Lewis for useful conversations, and S. L. Hu for supplying plasmids. Y-T. Y. was supported by a Damon RunyonWalter Winchell Cancer Fund Fellowship, DRG-676. This work was supported by NIH grant GM 28983 to J. L. M. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked bdverfisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received February 3, 1986; revised March 27, 1986.

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