in vitro transcription from the adenovirus 2 major late promoter

T H E .JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc

Vol. 259, No. 13, issue of July 10, pp. 8513-8521,1984 Printed in U. S. A.

In Vitro Transcription from the Adenovirus 2 Major Late Promoter Utilizing Templates Truncated at Promoter-proximal Sites*

(Received for publication, January 13, 1984)

Richard Jove and James L. ManleyS From the Department of Biological Sciences, Columbia Uniuersity, New York, New York 10027

We report that adenovirus 2 DNA sequences located between positions -66 and -51 upstream of the major late cap site (position +1) enhance transcription initiation from this promoter by up to 5- to 10-fold in HeLa whole cell lysates. This enhancing effect is template concentration-dependent and is abolished by truncation of the template immediately upstream of -66. Additionally, specific transcripts are not detected from templates truncated at +33 downstream of the cap site. These results define a minimum region of approximately 100 base pairs encompassing the transcription start site that appears to interact with the RNA polymerase 11 transcription complex during initiation.

Analysis of the shortest runoff transcripts that can be synthesized in uitro revealed that RNAs as short as 50 nucleotides are quantitatively modified by guanylylation and methylation to cap 1 structures. In contrast, short RNAs containing guanylylated but unmethylated cap structures are not efficiently utilized as substrates by endogenous cap-methylating enzymes in the HeLa lysate. These findings, together with the observation that the synthesis of short transcripts is sensitive to the presence of the methyltransferase in- hibitor ~-aden~ylhomocysteine, suggest that cap formation is a promoter-proximal event that occurs concomitantly with the synthesis of a nascent RNA polymerase I1 transcript.

The ability to reproduce faithfully the biogenesis of eukaryotic mRNA in soluble in vitro systems represents an important advance toward understanding the molecular events underlying eukaryotic gene expression. The synthesis and processing of an RNA polymerase I1 transcript to produce a mature mRNA is a multiple step process involving complex interactions among proteins and nucleic acids at every step. These steps include transcription initiation, elongation, termination, and processing of the nascent transcript by capping of the 5’ terminus, splicing together of noncont i~ous exon regions, and formation of a polyadenylated 3‘ terminus (1). The development of soluble in uitro transcription systems in which well defined DNA templates are accurately transcribed (2, 3 ) has facilitated rapid progress in identifying the control regions upstream of eukaryotic genes that direct transcription initiation by RNA polymerase I1 (1). A region containing the “TATA box”, a highly conserved sequence located approximately 25 to 30 base pairs upstream from the cap site of most

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of National Institutes of Health Grant GM 28983 in support of this work.

...._____ ~ _ _

mRNA-encoding genes (4), has been shown to be required for specific in uitro transcription of almost all genes examined that contain this sequence (5-8). A second region, located a variable distance farther upstream, also has been found to be required for efficient transcription in vitro of several genes examined to date (9-11). These two regions, therefore, appear to constitute distinct promoter elements that are recognized by RNA polymerase I1 in uitro. The feasibility of reproducing RNA processing events in soluble in uitro systems has also been demonstrated (12-14).

We have been particularly interested in investigating the molecular interactions that occur during transcription initiation by RNA polymerase 11. We have addressed questions related to promoter-proximal events by utilizing DNA templates truncated immediately upstream or downstream of the transcription start site as substrates for RNA polymerase I1 in a soluble in uitro transcription system. With this approach, we have investigated transcription initiation from a well stud- ied eukaryotic promoter, the Ad2’ major late promoter (5, 6, 11), and formation of the 5”terminal cap structure (15), a processing step that appears to be an extremely early event in the synthesis of a nascent transcript (16-18). Here we report evidence suggesting that the RNA polymerase I1 transcription initiation complex interacts with approximately 100 base pairs of template DNA surrounding the major late promoter, including a region upstream of the TATA box. We also present further evidence suggesting that cap formation occurs near the time of transcription initiation and is linked to the synthesis of nascent RNA polymerase I1 transcripts.

MATERIALS AND METHODS

R e c o r n b i ~ n ~ DNA-The Ad2 major late promoter pBR322 recombinants pXB210, pXB806, and p‘P4 have been described (6). Plasmid DNA was amplified and purified according to standard protocols (19). DNA templates were prepared for in uitro transcription by cleavage with the appropriate restriction endonucleases (New England Bio- labs), followed by phenol and chloroform extractions, and ethanol precipitation. Bacteriophage MI3 recombinants containing the de- sired Ad2 strands were constructed by inserting the Ad2 sequences of pXB2lO or p94 between the EcoRI and Hind111 restriction sites of the M13mp9 vector (20). Single-stranded DNA was prepared from phage as described (20) and further purified by additional phenol and chloroform extractions before use as hybridization probes.

HeLa Cell Extracts and in Vitro Transcription-HeLa whole cell lysates were prepared according to the method previously described (31, except that the final dialysis buffer was 40 mM Tris-C1, pH 7.9, 100 mM KCl, 10 mM MgClz, 0.1 mM EDTA, 2 mM dithiothreitol, 17% glycerol. Standard 25-pl reaction mixtures, containing 15 pl of cell extract, were 24 mM Tris-C1, pH 7.9, 60 mM KCl, 6 mM MgC12, 0.06 mM EDTA, 1.2 mM dithiothreitol, 10% glycerol, 4 mM creatine phosphate, and 1.25 pg of template DNA (except where otherwise indicated). Ribonucleoside triphosphates were present a t 50 p~ each,

The abbreviations used are: Ad2, adenovirus 2; AdoHcy, S-aden- osylhomocysteine; AdoMet, S-adenosylmethionine; MLP, major late promoter.

- -

8513

85 14 In Vitro Transcription from the Ad2 Major Late Promoter except for the labeled nucleotide, which was 10 p~ containing 5 to 200 pCi of [n-"PIGTP or [n-32P]CTP (New England Nuclear). AdoHcy and AdoMet (Boehringer Mannheim) were added to reaction mixtures as indicated in the text. Reaction mixtures were incubated at 30 "C for 1 h.

Analysis of RNA Transcripts by Gel Electrophoresis-RNA from in vitro transcription reactions was purified as described (21). RNAs shorter than 200 nucleotides in length were analyzed by fractionation through 10 or 12.5% polyacrylamide slab gels containing 7 M urea as described (19). followed by autoradiography of the wet gels with intensifying screens at -80 "C for 1 to 5 days. Samples to be analyzed by polyacrylamide/urea gel electrophoresis were ethanol precipitated, resuspended in sample buffer consisting of 90% formamide plus 0.2% sodium dodecyl sulfate, and heated to 90 "C for 2 min prior to loading. For the analysis of longer transcripts, purified RNA was glyoxalated and fractionated through 1.4% agarose gels according to the method of McMaster and Carmichael (22), followed by autoradiography of the dried gels without intensifying screens for 1 to 5 days. Autoradi- ograms were scanned with a Gilford model 250 densitometer.

RNA-DNA Hybridization and Ribonuclease Digestions-Purified "P-labeled RNA from a standard in vitro transcription reaction was treated with pancreatic DNase I (Boehringer Mannheim) as described (21). Hybridization of this RNA to an excess (at least 10-fold) of M13 single-stranded DNA recombinants containing Ad2 sequences complementary to the in uitro transcripts was performed in 5 X SSC (1 X SSC is 0.15 M NaCI, 0.015 M Na citrate) in a volume of 12 pi at 50 "C for 3 h. Nuclease digestions were carried out by the addition of 200 pl of ice-cold RNase digestion buffer (0.25 M NaCI, 50 mM Na acetate, pH 4.5, and 1 mM EDTA) with RNase T1 at 2.5 pg/ml, RNase T2 at 0.5 unit/ml, and RNase A at 2.5 pg/ml (Calbiochem). Digestion was at 25°C for 30 min, after which time the samples were extracted with phenol/chloroform (l:l), chloroform, and ethanol precipitated. RNA-DNA hybrids were denatured by heating in sample buffer (above), and the protected RNAs were resolved by electrophoresis through polyacrylamide/urea gels and visualized by autoradiography as described above.

Dihydroxyboryl Cellulose Chromatography and Fingerprint Analysis of Oligonucleotides-Purified RNA from a standard transcription reaction was digested with RNase T1, bound to a column of dihydroxyboryl cellulose (Collaborative Research), and eluted as described (16, 23). The oligonucleotides were resolved by two-dimensional fingerprint analysis, using homomixture C in the second dimension (16, 24).

Analysis of 5'-Terminal Cap Structures-RNA transcripts to be used for cap analysis were eluted from polyacrylamide/urea gel slices as previously described for the elution of DNA restriction fragments (25). RNase T2, P1 nuclease (Yamasa Shoyu Co.), and calf intestine alkaline phosphatase (Boehringer Mannheim) digestions were performed as described (26), and the digestion products were analyzed by high voltage DEAE-paper (DE81, Whatman) electrophoresis a t pH 3.5 in pyridine acetate (26). Appropriate cap core standards (P-L Biochemicals), visualized with ultraviolet light, and the dye markers xylene cyano1 FF and acid fuchsin were applied in parallel with the samples to be analyzed. For isolation of 5"terminal T1 oligonucleotides, purified RNAs were digested with RNase T1 (16), fractionated through thin (0.4-mm) 20% polyacrylamide slab gels containing 7 M urea (19). eluted, and purified. The T1 oligonucleotides were subjected to cap analysis as described above.

RESULTS

Transcription from Templates Truncated Upstream of the Cap Site-Previous studies have indicated that regions upstream of TATA box consensus sequences constitute additional elements of promoters recognized by RNA polymerase I1 in uiuo and in uitro (reviewed in Ref. 1). The upstream boundary of the Ad2 MLP sequences required for optimal transcription initiation in HeLa whole cell lysates has been shown to lie within the region between -66 to -51 (relative to the cap site, +l), even though the TATA box is located between -31 and -25 (6). To extend these studies, transcription initiation from the deletion mutants with MLP upstream boundaries at -66 or -51 was examined in more detail. A comparison of the levels of transcription from these two mutants in HeLa lysates revealed a striking template concen-

tration-dependent effect of MLP upstream sequences (Fig. 1, compare lanes a and 6 ) . At template concentrations of 50 pg/ ml or greater, transcription initiation from the -51 deletion mutant, pXB806, was reduced approximately 2-fold compared to the -66 deletion mutant, pXB210, as previously reported (6). In contrast, transcription initiation from the -51 deletion mutant was reduced by a factor of 5- to 10-fold compared to the -66 deletion mutant when the template concentration was 25 pg/ml. These results demonstrate that MLP upstream sequences located between -66 and -51 significantly enhance transcription initiation from this promoter and that this enhancement is template concentration-dependent in the HeLa lysate.

Since previous studies have demonstrated that MLP sequences upstream of -66 are not required for wild type levels of transcription in uitro (6), it was expected that truncation of the template immediately upstream of -66 would have no effect on transcription initiation. This prediction could be tested by examining transcription from the templates cleaved at these sites with EcoRI restriction endonuclease, as the deletion mutants used in these studies were constructed with EcoRI linkers inserted at the upstream boundaries of the Ad2

DNA u g h 1 : 25 50 100

a b c d a b c d a b c d M

pXB210L

pXB806T

a

-4.36 Kb

-1.43

-0.63

-0.37

FIG. 1. Effect of cleavage upstream of the Ad2 major late cap site on transcription initiation. Top, synthesis of AvaI runoff transcripts from the pXB210 and pXB806 templates, either uncleaved or cleaved with EcoRI at their respective Ad2 upstream boundaries, as a function of total DNA concentration (25, 50, or 100 pg/ml) in the HeLa whole cell lysate. The templates are: lanes a, pXB210 cleaved with AvaI; lanes 6, pXB806 cleaved wtih AvaI; lanes c, pXB210 cleaved with AuaI and EcoRI; lanes d, pXB806 cleaved with AuaI and EcoRI. Lune M: RNA size standards prepared by using p(d4 DNA cleaved with a variety of restriction enzymes as ternplates. Purified RNAs were glyoxalated and analyzed by electrophoresis in a 1.4% agarose gel. Bottom, positions of the relevant restriction sites in pXB210 and pXB806 relative to the major late cap site (+l). --, Ad2 sequences; - - -, pBR322 sequences. The 5' termini of the EcoRI cleaved templates are located 6 nucleotides upstream of the indicated Ad2 upstream boundaries (Ref. 6).

In Vitro Transcription from the Ad2 Major Late Promoter 8515

3 194-

81-

52-

33-

B

1 2 3 4

194-

52-

M

-220

-1 54

-75

FIG. 2. Effect of cleavage downstream of the Ad2 major late cap site on transcription initiation. A, lanes I , 3, 5, and 7, transcripts synthesized in standard transcription reactions using pXB210 cleaved with the indicated restriction endonucleases as tem-

sequences (6). Surprisingly, when the -66 deletion mutant was cleaved with EcoRI, the level of transcription obtained from this template was reduced to that obtained with the -51 deletion mutant at all template concentrations examined (Fig. 1, compare lanes a, b, and c). Thus, truncation of the template immediately upstream of -66 abolishes the transcription- enchancing ability of MLP upstream sequences located between -66 and -51. In contrast, cleavage of the -51 deletion mutant with EcoRI at its upstream boundary did not significantly reduce the level of transcription (less than 2-fold) from this template (Fig. 1, lanes b and d). This result argues against trivial explanations for the observed effect of truncation at -66 (such as proteins that bind DNA ends and thereby interfere with the transcription apparatus). The above results suggest that nonspecific template DNA upstream of -66 is required to stabilize an interaction between the transcription complex and an upstream region, located between -66 and -51, that enhances transcription initiation from this promoter.

Transcription from Templates Truncated Downstream of the Cap Site-Previous studies with MLP deletion mutants have indicated that specific DNA sequences downstream of the cap site are not required to obtain wild type levels of transcription from this promoter in HeLa whole cell lysates (6). T o determine the extent to which nonspecific template DNA beyond the cap site is required to support transcription initiation by RNA polymerase I1 in vitro, truncated templates were prepared by cleavage of pXB210 with restriction endonucleases that progressively delete more template downstream of the cap site (Fig. 2). Runoff transcripts of the predicted sizes were observed when pXB210 was digested with any one of the restriction endonucleases HindIII, DdeI, or HphI (which cleave a t +194, +81, and +52, respectively), and incubated in the whole-cell lysate (Fig. 2 A , lanes 1,3, and 5). Cleavage of the template with RsaI at +47 yielded results similar to cleavage a t +52 (data not shown). In contrast, no transcripts were detected when the template was cleaved a t +33 with PuuII (Fig. 2A, lane 7). The synthesis of the transcripts seen in lanes 1, 3, and 5 was completely abolished by the addition of cu-amanitin a t 0.5 pg/ml to the reaction mixtures, indicating that these transcripts were products of RNA polymerase I1 (Fig. 2A, lanes 2,4, and 6).

To confirm that these transcripts originated from the MLP and were not initiated at other nonpromoter sites (for exam- ple, the ends of DNA fragments or RNA polymerase I1 promoter-like sequences in the pBR322 plasmid vector), the RNAs were hybridized to the Ad2 sequences from pXB210 that had been recloned into an M13 bacteriophage vector and then treated with a mixture of ribonucleases (see "Materials and Methods") to digest unprotected RNAs. The single- stranded phage DNA probe used in this ribonuclease-mapping assay contains the Ad2 strand that is complementary to RNAs

determined from the nucleotide sequence of Ad2.2 Lanes 2, 4, 6, and 8, as above except that 0.5 pg/ml of a-amanitin was included in the reaction mixtures. Purified RNAs were resolved in a 12.5% polyacrylamide gel containing 7 M urea. B, ribonuclease mapping of transcripts shown in A above. Purified 32P-labeled RNA was hybridized to a single-stranded DNA probe containing the complementary Ad2 sequences of pXB210 (recloned into an M13 phage vector), digested with a mixture of RNases, and the protected RNAs were analyzed as described above. Lanes I , 2, and 3, protected RNAs from transcription reaction mixtures incubated with pXB210 cleaved with HindIII, DdeI, and HphI, respectively. Lane 4, minus probe control with RNA synthesized using pXB21O cleaved wtih HphI. Lane M, end-labeled TaqI digest of pBR322 used as size standards.

plates. The positions of the cleavage sites (bottom of figure) were * R. J. Roberts, personal communication.

8516 In Vitro Transcription from the Ad2 Major Late Promoter

transcribed in the correct direction relative to the promoter. The sizes of the protected RNAs in each case were identical to the sizes of the runoff transcripts (Fig. 2B, lanes 1 to 3) . The template cleaved with DdeI also gave rise to transcripts approximately 50 nucleotides long and to a heterogeneous propulation of transcripts ranging in size from approximately 70 to 80 nucleotides. These RNAs were also synthesized by RNA polymerase I1 and protected in the ribonuclease-mapping assay (Fig. 2 A , lanes 3 and 4, and Fig. 2B, lane 2). Evidence that all of these transcripts, observed only when pXB210 cleaved with DdeI was used as a template, were initiated at the correct in vivo transcription start site is presented below. These additional RNAs, therefore, most probably arose by abortive terminations a t discrete sites on this template.

The possibility was considered that the lack of discrete runoff transcripts from the template cleaved with PvuII was due to random abortive terminations by the polymerase, re- sulting in RNAs with heterogeneous 3' termini that would, therefore, not be detected by size fractionation through polyacrylamide/urea gels. To investigate this possibility, RNA extracted from an in vitro transcription reaction that had contained either the PvuII- or HindIII-cleaved template was digested to completion with RNase T1, chromatographed on dihydroxyboryl cellulose columns, and the oligonucleotides containing 2',3'-cis diols were resolved by two-dimensional fingerprint analysis. Fig. 3A (arrow) shows the capped 5'- terminal RNase T1-resistant undecanucleotide, indistin- guishable from the predominant in vivo major late capped T1 oligonucleotide (3, 23) that is synthesized in vitro using the HindIII-cleaved template. In contrast, no capped oligonucleotides are detected when the PvuII-cleaved template was used (Fig. 3B). This result, however, does not rule out the possibility that uncapped transcripts initiated at the correct start site but with heterogeneous 3' termini were synthesized, since only capped 5"terminal oligonucleotides containing 2',3'-cis diols are detected by this assay. With this caveat, these results strongly suggest that transcription initiation does not take place on templates truncated approximately 30 base pairs downstream from the cap site. The 33-nucleotide transcript also was not detected when the concentration of DNA ends in the reaction mixture was increased 10-fold by digestion of the PvuII-cleaved template with HphI and DdeI (data not shown), arguing against the possibility that this result is due to interference from proteins that bind DNA ends. These observations suggest an interaction between some component of the RNA polymerase I1 transcription complex and sequences extending to a region between 30 and 50 base pairs downstream from the transcription start site during initiation.

5"Terminal Cap Structure of Short Runoff Transcripts- Runoff transcripts synthesized in the whole cell lysate from the MLP promoter have been shown to be modified at their 5' termini by guanylylation and methylation (3). Previous in vitro and in vivo data suggest that formation of the cap structure is an extremely early event in the synthesis of a nascent RNA polymerase I1 transcript (16, 17). I t was, therefore, of interest to determine whether the 5' termini of the very short in vitro runoff transcripts are also modified by guanylylation and methylation. As shown in Fig. 3A, transcripts 190 nucleotides long contain an RNase T1-resistant 5"terminal oligonucleotide that can be selected by dihydroxyboryl cellulose chromatography, indicating that these transcripts are capped.

To examine the 5' termini of the shortest detectable in uitro runoff transcripts (50 nucleotides), RNA synthesized using pXB210 cleaved with HphI was fractionated by poly-

A . e

0 0

B

i4- 1 I

FIG. 3. Two-dimensional fingerprint analysis of capped 5'- terminal oligonucleotides synthesized using pXB2 10 templates. Purified RNA from a standard transcription reaction was digested with RNase T1, and the capped oligonucleotides were selected on a dihydroxyboryl cellulose column and resolved by two- dimensional fingerprint analysis. Electrophoresis in the first dimension was from right to left, and homochromatography in the second dimension was from bottom to top. A, RNA from a reaction mixture incubated with pXB210 cleaved with HindIII. Arrow, the predominant capped T1 undecanucleotide found at the 5' termini of Ad2 major late transcripts. B, RNA from a reaction mixture incubated with pXB210 cleaved with PuuII.

acrylamide/urea gel electrophoresis, eluted from gel slices, subjected to a series of nuclease digestions, and analyzed by high voltage DEAE-paper electrophoresis a t pH 3.5 in pyridine acetate. P1 nuclease digestion of this RNA yielded a single P1-resistant product that comigrates with authentic 7mG(5')pppAm marker (Fig. 4, lane 2). When the transcription reaction was carried out in the presence of 50 p~ AdoHcy, the predominant P1-resistant product comigrated with authentic G(5')pppA marker (Fig. 4, lane 1). Since P1 nuclease cleaves adjacent to a 2"O-methyl group, RNase T2 and T2 plus alkaline phosphatase digestions were performed to further characterize the PI-resistant structures (Fig. 4, lanes 3 to 6). These analyses are consistent with the presence of a 2'- 0-methyl at the penultimate residue, in addition to the 7- methylguanosine, in all of the capped 5' termini. From the pattern of these results and the electrophoretic migration of the RNase T2 plus alkaline phosphatase digestion products relative to the dye markers acid fuchsin and xylene cyano1 FF in this system (26), the structure at the 5' termini of the methylated transcripts was deduced to be cap I. In no case has evidence for the formation of cap I1 structures in the whole cell lysate been obtained. From the known sequence at


-7mCpppAm

.CPPPA

-7mCpppAmpC

3 4 5 6

FIG. 4. Analysis of cap structures at the 5’ termini of short runoff transcripts. Runoff transcripts approximately 50 nucleotides long synthesized from the template pXB210 cleaved with HphI were fractionated in a polyacrylamide/urea gel, eluted from gel slices, digested with specific nucleases, and the products were resolved by high voltage DEAE-paper electrophoresis a t pH 3.5. The digestions were: lanes I and 2, P1 nuclease; lanes 3 and 4, RNase T2 plus alkaline phosphatase; lanes 5 and 6, RNase T2. The RNA was synthesized in the presence of [cx-~’P]GTP in standard transcription reactions either with (lanes 1,3, and 5) or without (lanes 2,4, and 6 ) 50 PM AdoHcy. The appropriate cap core standards, visualized with ultraviolet light, were applied in parallel. 0, origin.

the 5’ terminus of the Ad2 major late transcript (16), the 5’ terminal structure corresponds to ‘mG(5’)pppAmpCp. These results demonstrate that RNAs as short as 50 nucleotides in length, transcribed by RNA polymerase I1 in the HeLa lysate, are modified by methylated cap structures at their 5’ termini.

The results shown in Fig. 4 raised the possibility that the extent of capping of the 50-nucleotide runoff might be less than complete, based on the estimated ratio of 7mG(5‘)ppp- Am to pG (from the nucleotide sequence, the expected ratio is 1:16). The possibility was considered that this result might indicate the presence of uncapped ends such as pppA, which would not be labeled with [a-“PIGTP and, therefore, not detected. Alternatively, this apparent ratio bias may have arisen from background contamination of unrelated RNA species that were labeled in the lysate and copurified with the 50-nucleotide runoff. Therefore, in order to assess more accurately the extent of capping of this runoff transcript, the labeled RNA corresponding to approximately 50 nucleotides was purified from reaction mixtures that had contained HphI- cleaved pXB210 and [a-“PICTP. The major late 5’-terminal T1 undecanucleotide was then isolated by digestion of this RNA with RNase T1 and fractionation of the products through a 20% polyacrylamide/7 M urea gel. As shown in Fig. 5A, an oligonucleotide of the expected size for the 5”terminal

XCFF-

-CP

a -11

-GP

0 -0

FIG. 5. Purification and analysis of major late 5‘4erminal T1 oligonucleotide. A, 50-nucleotide-long runoff RNA, synthesized in the presence of [cx-~*P]CTP and purified as described in Fig. 4, was digested to completion with RNase TI , and the resultant products were fractionated in a 20% polyacrylamide/urea gel. The largest expected T1 product from this runoff is the major late 5”terminal undecanucleotide (indicated by Tl ) ; the next largest expected T I product is 7 nucleotides. Marker dyes used as size standards (Ref. 19): XCFF, xylene cyano1 FF (28 nucleotides); BPB, bromophenol blue (8 nucleotides). B, RNase T2 digest of purified 5“terminal TI undecanucleotide analyzed by high voltage DEAE-paper electrophoresis. 0, origin. C, structure of capped major late 5”terminal T1 oligonucleotide and the expected ratios of labeled RNase T2 digestion products. Dots indicate phosphates labeled when transcription is carried out in the presence of [cx-~’P]CTP.

fragment resulted from T1 digestion of the [~r-~*P]cTP-labeled RNAs. This oligonucleotide was purified, digested with RNase T2, and subjected to high voltage DEAE-paper electrophoresis as described above.

The results of this analysis, shown in Fig. 5B, are consistent with complete capping of the 5”terminal T1 undecanucleotide. This conclusion is based on visual inspection of the autoradiogram and comparison with the results expected for complete capping of the 5”terminal oligonucleotide, shown in Fig. 5C (it was not possible to obtain sufficient counts in any of the cap analyses to permit direct quantitation of radioactivity in each spot). Examination of the autoradiogram in Fig. 5B could lead to the suggestion that the relative intensity of the spot corresponding to the cap structure is greater than expected for complete capping. This is attributed to the fact that the highly charged cap structures diffused less than the nucleotides during the electrophoresis on DEAE- paper, consistent with our previous observations (3). Two additional discrete bands are observed in Fig. 5A that resulted from T1 digestion of the [c~-~~P]CTP-labeled RNA. The origin of these RNAs is not known; however, the RNase T2 digestion products are not related to those of the major late 5”terminal T1 undecanucleotide (data not shown). Furthermore, only a single species of the size expected for the 5”terminal T1 oligonucleotide was obtained from RNAs labeled with either [~Y-“~P]CTP or [~Y-~’P]GTP (data not shown). Finally, we have not detected any evidence of 5‘ triphosphate termini in RNase T2 digests of [a-3ZP]CTP-labeled RNA (the pppAp product would be labeled in this case). The above results, therefore, strongly suggest that RNAs as short as 50 nucleotides, transcribed by RNA polymerase I1 from the MLP in HeLA lysates, are completely modified by guanylylation and methylation to cap I structures.

8518 In Vitro Transcription from the Ad2 Major Late Promoter

Post-transcriptional Methylation of G(5’)pppA Termini- The finding that short runoff transcripts are capped and methylated raised the question of whether cap formation occurred before or after release of the nascent transcripts by the polymerase. To address this question, the following ex- periment was performed to determine whether guanylylated but unmethylated 5’ termini of short RNAs are post-tran- scriptionally methylated in the HeLa lysate. RNA was synthesized in the presence or absence of 50 p~ AdoHcy using pXB210 cleaved with DdeI, which gives rise to multiple transcripts that originate from the MLP (Fig. 2). RNA from three different size classes (Fig. 6A), corresponding to approximately 50, 70, or 80 nucleotides in length, was purified, and the 5”terminal cap structure of each size class was analyzed as described above (the yield of the 80-nucleotide species was, unexpectedly, greatly reduced in the presence of 50 p~ AdoHcy, see below). The 5’ termini of RNAs in all these size classes contained cap 1 structures when transcription was carried out in the absence of AdoHcy (Fig. 6B, lanes a, b, and c ) . In the presence of AdoHcy, the 5’ termini in all size classes consisted of G(5’)pppA (Fig. 6B, lanes d, e, and f). These guanylylated but unmethylated RNAs were then reincubated in the lysate under standard conditions without AdoHcy, fractionated by polyacrylamide/urea gel electrophoresis, and RNA in the 50-nucleotide or the combined 70- to 80-nucleotide range purified and analyzed as above. As shown in Fig. 6C (lanes 4 and 6), a large fraction of the G(5’)pppA termini remained unmethylated. As a control, RNAs containing cap I structures were unchanged following reincubation in the lysate (Fig. 6C, lanes 3 and 5).

B 4

1 2

- dC b l t c

OC I C

a

C @

1

b c

.

4

2 3

d e

4 5

FIG. 6. Efficiency of post-transcriptional methylation of guanylylated 5‘ termini. A, transcripts synthesized from pXB210 cleaved with UdeI in the absence (lane 1 ) or presence ( l a n e 2) of 50 PM AdoHcy resolved by polyacrylamide/urea gel electrophoresis. Re- gions labeled a-f indicate transcripts isolated and analyzed below. The approximate sizes of these RNA species, from top to bottom, are 80,70, and 50 nucleotides. B, isolated RNAs from above were digested with RNase T2 plus alkaline phosphatase and analyzed by high voltage DEAE-paper electrophoresis. Lanes a-f, RNA species corresponding to regions of gel indicated in A above. C, post-transcriptional methylation of guanylylated 5’ termini. RNAs from A were reincubated in the lysate under standard conditions without AdoHcy, purified by polyacrylamide/urea gel electrophoresis, digested with P1 nuclease, and subjected to high voltage DEAE-paper electrophoresis. The reincubated RNAs were: the combined 70 to 80-nucleotide species that had originally contained methylated ( l a n e 3) or unmethylated ( l a n e 4 ) 5’ termini and the 50-nucleotide species previously methylated (lane 5 ) or unmethylated (lane 6). Lanes 1 and 2, markers for methylated and unmethylated cap structures, respectively.

The above results indicate that post-transcriptional methylation of guanylylated 5’ termini is inefficient compared to the essentially complete formation of methylated cap I structures observed when transcription is carried out in the absence of AdoHcy. This finding suggests that formation of the cap structure occurs concomitantly with the synthesis of a nascent transcript under the standard transcription conditions in the lysate. I t is interesting that a larger percentage of the RNA 50 nucleotides long is methylated (up to approximately 50%) compared to RNAs in the 70- to 80-nucleotide range (Fig. 6C, compare lanes 4 and 6). The finding that shorter transcripts are better substrates for the endogenous cap-methylating enzymes is consistent with the suggestion that nascent transcripts are capped at a very early time after transcription initiation.

Effect of AdoHcy on the Synthesis of Short Runoff Tran- scripts-Previous studies have demonstrated that AdoHcy prevents the accumulation of RNA polymerase I1 transcripts in vitro when present at high concentrations (greater than 250 p ~ ) during transcription initiation but has no effect on the elongation of long runoff transcripts, suggesting a close association between the transcription complex and capping enzymes during initiation (18). When the synthesis of short runoff transcripts from DdeI-cleaved pXB210 was examined, however, the synthesis of a subpopulation of transcripts, ranging in size from approximately 75 to 80 nucleotides, was found to be hypersensitive to AdoHcy relative both to transcripts approximately 50 and 70 nucleotides long (Fig. 6A), as well as to transcripts much longer (e.g. Ref. 18). This observation cannot be explained by differences in cap structures that might result in altered electrophoretic mobilities, since the RNAs in all of these size classes contain identical cap structures (Fig. 6B). These results raised the possibility that the presence of AdoHcy promotes abortive termination before the polymerase has reached the end of the template. Alter- natively, it was possible that the polymerase initiates a t more than one site on this template and that initiation at different sites may be differentially sensitive to AdoHcy.

To investigate these possibilities, the DdeI-cleaved pXB210 template was transcribed in the presence of increasing concentrations of AdoHcy, from 50 p~ to 1 mM (Fig. 7A, lanes 1 to 4) . The RNA synthesized was then hybridized to a single- stranded M13 phage DNA probe containing complementary Ad2 sequences extending downstream of the cap site to position +33, and the RNase-resistant products were resolved by polyacrylamide/urea gel electrophoresis. A single RNA species, approximately 33 nucleotides in length, was protected by the single-stranded DNA probe and was not present in the minus probe control (Fig. 7B, lanes 1 to 4) . Furthermore, the relative intensities of these bands are identical whether transcription was carried out in the presence or absence of 50 p~ AdoHcy, but reduced in the presence of 1 mM AdoHcy (compare lanes 1, 2, and 3) . These results show that all of the transcripts observed initiated at the correct start site and that the effect of AdoHcy a t low concentrations must be to promote premature termination(s) near the truncated end of the template. Thus, elongation of nascent transcripts by RNA polymerase I1 downstream of +33 near the ends of the short truncated templates is hypersensitive to AdoHcy, perhaps as a result of an altered conformation of the cap methylases in the transcription complexes in the presence of AdoHcy. Such an altered conformation might render the transcription complexes unstable and thereby promote abortive terminations. At high AdoHcy concentrations, however, transcription by RNA polymerase I1 was inhibited before the nascent transcript had elongated 33 nucleotides (Fig. 7B, compare lanes 1


A

1 2 3 4 5 6

81,

50,

B 1 2 3 4 5

-81

-50

AMICY: a05 a s 1.0 01)s m: I I I I IwJ&

I d

FIG. 7. Effect of AdoHcy on the synthesis of short runoff transcripts. A, lanes 1 to 6, transcription products from reaction mixtures incubated with pXB210 cleaved with DdeI and increasing concentrations of AdoHcy and/or AdoMet (as indicated in figure). Purified RNA was analyzed as described in Fig. 2. Most of the higher molecular weight bands, which were insensitive to AdoHcy, originated from end-labeling of template DNA fragments in the lysate, as these were eliminated by DNase I digestion (see lane 5 below). E, 32P- labeled RNA from A was hybridized to a single-stranded DNA probe containing major late sequences extending to +33, digested with a mixture of ribonucleases, and the protected RNAs were analyzed as described in Fig. 2. Lanes 1, 2, and 3, protected RNAs from reaction mixtures containing 0,50 pM, or 1 mM AdoHcy, respectively. Lane 4, minus probe control using RNA from a standard reaction mixture without AdoHcy. Lane 5, RNA from a standard reaction mixture, after digestion with DNase I, not treated with ribonucleases. Arrow, position of protected RNA approximately 33 nucleotides long.

and 3). The above results, therefore, suggest that the enzymes that catalyze cap formation are associated with promoter- proximal RNA polymerase I1 transcription complexes.

AdoHcy inhibits the activity of many methyltransferases by competing with the methyl group donor, AdoMet, for binding to the enzyme-active site (27). In theory, therefore, it might be expected that the inhibition of transcription by AdoHcy could be “rescued by the addition of enough exoge- nous AdoMet to compete out the AdoHcy. However, at the high AdoHcy concentrations that are most effective in inhib- iting transcription by RNA polymerase I1 in uitro (greater than 250 p ~ ) , the addition of AdoMet does not rescue the inhibition? The difficulty in interpreting this result is that the addition of AdoMet to concentrations greater than 1 mM nonspecifically inhibits all transcription (data not shown). Thus, if the cap methylases have a lower K,,, for AdoHcy than for AdoMet, the rescue by very high concentrations of AdoMet would be impossible to demonstrate. The finding reported here, that the synthesis of short transcripts is partially inhibited at much lower AdoHcy concentrations, allowed the possibility of testing for rescue at AdoMet concentrations that do not nonspecifically inhibit transcription. In fact, when pXB210 cleaved with DdeI was incubated in a reaction mix-

R. Jove and J. L. Manley, unpublished results.

ture with 50 p~ AdoHcy plus 250 p~ AdoMet, the synthesis of transcripts 75 to 80 nucleotides was restored to the levels observed in the absence of AdoHcy (Fig. 7A, lanes 1 and 5). This observation establishes that the inhibition of RNA polymerase I1 transcription is mediated directly by AdoHcy and not a metabolite or contaminant of AdoHcy.

DISCUSSION A number of novel results have been obtained by utilizing

templates truncated upstream or downstream of the Ad2 major late cap site as substrates for RNA polymerase I1 in HeLa whole cell lysates. MLP upstream sequences located between -66 and -51 increase the level of transcription initiation from this promoter by up to 5- to 10-fold in a template concentration-dependent manner. Truncation of the template near -66, however, abolishes this transcription- enhancing effect, perhaps by destabilizing the binding of some component of the transcription complex to MLP upstream sequences during initiation. In contrast, truncation of the template near -51 has little effect on the ability of sequences located downstream of -51 to promote transcription initiation. It is significant that the requirement for the upstream region is partially overcome at high template concentrations, indicating that under this condition the level of transcription is determined primarily by MLP sequences downstream of -51. Although the molecular basis for these findings is not clear, it appears that two distinct interactions take place during transcription initiation from the MLP. One interaction involves the region upstream of -51 and the other the region downstream of -51, containing the TATA box. These observations suggest that the upstream region constitutes a promoter element that is functionally distinct from the TATA box.

It has recently been reported that sequences located between -97 and -34 are required for efficient expression of the MLP in vivo and in uitro (11). These investigators pro- posed that an MLP sequence located between -69 and -59, which shares strong homology with a rabbit @-globin upstream sequence centered around -78 that has been shown to be important for the in uiuo expression of that gene (28), may be responsible for the observed upstream effect. The results reported here, together with the observation that specific MLP sequences upstream of -66 are not required to obtain wild type levels of transcription in uitro (6), are consistent with the suggestion that this upstream homology is a func- tional element of the MLP. This conclusion is further sup- ported by experiments indicating that MLP sequences upstream of -66 are not required for optimal expression of this promoter in uiuo, whereas sequences located between -66 and -59 are.‘ Sequences upstream of the TATA box have also been shown to be required for efficient expression of several other eukaryotic promoters in uitro and in vivo (1). The results from this study also demonstrate that DNA ends cannot substitute for MLP upstream sequences in uitro, as previously suggested in the case of another RNA polymerase I1 promoter (10). This argues against the suggestion that DNA ends can act as “entry sites” for RNA polymerase 11, thereby eliminat- ing the need for upstream sequences. Interestingly, results analogous to those reported here utilizing truncated templates have also been observed with prokaryotic systems; cleavage of templates containing RNA polymerase promoters within the upstream -35 homology region inactivates these promoters in uitro (29).

When the MLP template is cleaved downstream of the cap site, specific runoff transcripts are detected when the template

‘ D. Lewis and J. L. Manley, manuscript in preparation.

In Vitro ~ r a ~ ~ c r i ~ t i o n from the Ad2 Major Late Promoter

is truncated at +52, but not at +33, suggesting that the transcription complex interacts with the template in this region during initiation. This interaction appears to be nonspecific, since previous studies indicate that Ad2 sequences downstream of the cap site are not required for accurate and efficient initiation i n vitro (6). An interesting analogy can also be made here with prokaryotic systems; pancreatic DNase digestion of templates containing promoter regions bound in stable complexes with prokaryotic RNA polymerase yields a protected DNA fragment extending as far as 20 base pairs downstream of the transcription start site, suggesting an interaction between the polymerase and this region that appears to be nonspecific (30-32). The RNA polymerases in the DNase-resistant complexes can synthesize short runoff transcripts, indicating that these are initiation complexes. This precedent suggests that it is reasonable to postulate that some part of the HeLa RNA polymerase I1 transcription complex bound to the promoter region extends out some 30 or so base pairs beyond the transcription start site. Perhaps cleavage of the template at +33 prevents formation of a stable initiation complex, thus accounting for the apparent inability of this template to support transcription initiation.

Analysis of the shortest runoff transcripts that we have detected i n uitro (50 nucleotides~ revealed that the 5’ termini of these RNAs were fully modified by guanylylation and methylation to cap I structures. In contrast, post-transcriptional methylation of guanylylated 5’ termini was relatively inefficient when this processing step was uncoupled from the synthesis of a nascent transcript. This finding suggests that, under the standard i n vitro transcription conditions, cap formation occurs before release of the nascent transcripts by the polymerase. These observations are consistent with previous suggestions that cap formation is an extremely early event that is linked to the synthesis of an RNA polymerase I1 transcript (16-18). Such a link would account for the observation that RNA polymerase I1 transcripts are specifically and completely capped, while RNA polymerase I and I11 transcripts, whether synthesized in vivo or in soluble i n vitro systems (33): are not capped at all. This specificity could also be explained by postulating that the capping enzymes can distinguish between the different RNA classes by recognizing unique features at the 5’4erminals (such as a specific recog- nition sequence); however, this possibility is extremely un- likely because the only known feature that distinguishes the 5”terminals of mRNAs from other RNA classes is the cap structure itself. The precise step during which cap formation occurs remains to be determined. It has recently been reported that very short abortive transcripts, less than 20 nucleotides in length, synthesized under the condition of limiting ribonucleoside triphosphate concentrations in vitro, are not yet capped (34). Thus, the available evidence suggests that cap formation is an event that takes place sometime after transcription initiation but before the nascent transcript has reached a length of 50 nucleotides. This conclusion is consistent with the postulated link between capping and RNA polymerase I1 transcription, since cap formation would then be expected to be a promoter-proximal event.

We have shown previously that the synthesis of RNA polymerase I1 transcripts is specifically inhibited when AdoHcy is present during transcription initiation and that elongation of long runoff transcripts is not detectably affected by this compound (18), suggesting that the capping enzymes are associated with the polymerase at the time of initiation. However, the observation reported here that the elongation _. “

R. Tjian, personal communication.

j~////////l1/1lt////////////l.~ “ - - ”2“ ””. I I I ! I 1 I

-66 -51 -31 -25 t1 i30 *so I L_ -

Upstream TATA Cap Region Box Formation

FIG. 8. A h ~ t h e t ~ e ~ ~llustration of the RNA polymerase TI transcription complex bound to the Ad2 major late promoter at initiation. Regions of the template postulated to interact with promo~r-bound tr~scription complexes are represented by the thick line. Hatched area indicates regions of specific interactions between promoter sequences, including the TATA box and an upstream region, and the transcription complex. Cap formation, catalyzed by enzymes associated with the complex, occurs before the nascent transcript (represented by the wavy line) has elongated 50 nucleotides. Coordi- nates are relative to the major late cap site (position +l).

of transcripts by polymerases near the truncated ends of short templates is h~ersensitive to AdoHcy suggests that cap methylases are at least transiently associated with promoter- proximal polymerases at very early times after transcription initiation. This suggested association between capping enzymes and the transcription complex provides a molecular mechanism for establishing a link between the synthesis of an RNA polymerase I1 transcript and cap formation, The fact that animal viruses with virion-associated RNA polymerases also code for virion-capping enzymes (35) is consistent with the hypothesis that the RNA polymerases interact with the capping enzymes in a specific manner. The postulated link in the case of mammal~an RNA polymerase I1 is not obligatory, however, since transcripts containing G(5’)pppA at their 5‘ termini are synthesized in the presence of AdoHcy, as previously observed (36). Other lines of evidence also indicate that transcription and cap formation are not obligatorily linked in this system (36,37).

The results discussed above are summarized in the model illustrated in Fig. 8. The RNA polymerase I1 transcription complex undoubtedly consists of many subunits but is represented here as a “black box” because the components com- prising a complete complex remain to be elucidated. The novel feature of this model is the suggestion that components of the transcription complex cover a region of approximately 100 base pairs of template DNA encompassing the Ad2 MLP during initiation. In this region, specific Ad2 sequences that comprise elements of the promoter are located within 70 base pairs upstream of the cap site. Two distinct promoter elements are located in this region, the TATA box and an upstream region. Downstream of the cap site, the transcription complex bound to the promoter interacts nonspecifically with template DNA extending 30 base pairs or more beyond the transcription start site. Formation of the 5”terminal cap structure, catalyzed by enzymes associated with the polymerase, occurs before the transcript has elongated 50 nucleotides. More refined and extensive analyses will be required, however, before the complex details of the molecular interactions and events underlying this simple model can be evaluated.

A c k n o ~ ~ d g ~ n ~ - W e thank B. Jacober for expert secretarial as- sistance.

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in vitro transcription from the adenovirus 2 major late promoter

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