changing the rna polymerase specificity of u snrna gene promoters

Cell, Vol. 55, 435-442, November 4, 1988, Copyright 0 1988 by Cell Press

Changing the RNA Polymerase Specificity snRNA Gene Promoters

lain W. Mattaj: Nina A. Dathan,” Huw D. Parry, Philippe Carbon,? and Alain Krolt * European Molecular Biology Laboratory Meyerhofstrasse 1 8900 Heidelberg, Federal Republic of Germany t Laboratorie de Biochimie lnstitut de Biologie Moleculaire et Cellulaire du CNRS 15 Rue Descartes 67084 Strasbourg Cedex, France

Summa

The promoter of a Xenopus tropicalis U6 gene can be transcribed by both RNA polymerases II and Ill. Two distinct elements, a TATA-like sequence and the region of transcription initiation, are only required for transcription by RNA polymerase Ill, while further common elements are required for transcription by both polymerases. Based on the unusually stringent requirement for a purine at the normal position of polymerase Ill transcription initiation and on the properties of mutants in this region, we suggest that RNA polymerase Ill itself may recognize the site of transcription initiation and thus be directly involved in efficient promoter selection. We have used the information obtained on U6 promoter structure to manufac- ture a U6 promoter that is RNA polymerase II-specific and to change the Xenopus U2 gene promoter specificity from RNA polymerase II to RNA polymerase Ill.

fntroduction

Of the major vertebrate U snRNAs, Ul-U5 are transcribed by RNA polymerase II (pol II) while U6 is transcribed by RNA polymerase Ill (pol Ill) (Dahlberg and Lund, 1987). Extensive studies of the pol II class of promoters have revealed that all contain at least two common elements, the distal sequence element (DSE) or enhancer and the prox- imal sequence element (PSE). Functional analysis of DSEs has shown that they are not identical (Dahlberg and Lund, 1987; Ares et al., 1987; Kazmaier et al., 1987; Hoff- man et al., 1986, and G. Tebb and I. W. Mattaj, submitted) but that they share some properties such as their almost constant position, roughly 250 bp upstream from the initiation site, and their possession of at least one octamer motif (ATGCAAAT), which is combined with different transcription factor binding sites in different DSEs. The PSE, which lies between 40 bp and 70 bp upstream from the initiation site, is essential for transcription, specifies the position of transcription initiation, and has been found so far only in the promoters of small RNA genes, although not only in U snRNA genes (Dahlberg and Lund, 1987; Mur- phy et al., 1987).

Although U6 is transcribed by RNA polymerase Ill (Kunkel et al., 1986; Reddy et al., 1987; Krol et al., 1987),

its promoter has striking similarity to the poi II class of U snRNA gene promoters (Krol et al., 1987). This extends to having a functional DSE that contains an octamer motif (Bark et al., 1987; Carbon et al., 1987; Das et al., 1988; Kunkel and Pederson, 1988) and an essential PSE-like sequence (Carbon et al., 1987; Kunkel and Pederson, 1988). Unlike other pol Ill genes, U6 does not contain gene internal sequences downstream of +l that are either essential for transcription or that affect the efficiency of transcription from the U6 promoter (Das et al., 1988).

Since all the sequence elements described so far in the U6 promoter are similar to those found in pof II promoters, we undertook a mutational analysis to determine which sequences are responsible for conferring pot Ill specificity on the U6 gene. Two elements, a TATA-like sequence and the sequence at and immediately surrounding the initiation site, are shown to be required for poi 111 transcription. Unexpectedly, we have discovered that the U6 promoter is transcribed both by pol II and pol Ill, We have used this information to construct simple mutants that result in the production of a pol II-specific U6 promoter and a U2 promoter with pol III specificity.

Results

Evidence That Pol 111 May Interact With the Initiation Region Sequences downstream of +l of the coding region are not required for U6 gene transcription (Das et al., 1988). Dur- ing our preliminary studies, however, we noted that altera- tions to and deletions in the sequence near +I could have dramatic effects on promoter efficiency (data not shown). The results of a detailed analysis of point mutations in this region are shown in Figure 1. A series of single and double point mutants, whose sequences are listed at the bottom of Figure lA, were first coinjected with a 148 maxigene (U6M), which has a wild-type promoter but which gives rise to elongated transcripts due to an insertion in its coding sequence (Carbon et al., 1987). The evidence that the U6 transcripts are made by RNA polymerase Ill is based on their a-amanitin sensitivity, the fact that their transcription is sensitive to competition by coinjected 5s and tRNA genes, and the fact that their coding region ends in a run of T residues, which is essential for their termination (Car- bon et al., 1987; Das et al., 1988, unpublished data). Throughout the paper, this assay is used purely to analyze transcription by RNA polymerase III. The sequence TCGT is the wild-type initiation region, and G is the initiation site. Alteration of this G to C decreases transcription (Figure lA, lane 2) by a factor of at least 20-fold (Figure IB, compare lanes 1 and 2).

Two conclusions can be drawn from the data in Figure 1A. The U6 promoter has an extreme dependence on a guanosine residue located at position +1 (in Figure lA, lanes i, 3,4,6,8, and 9 have a guanosine at +1 and are transcribed efficiently; lanes 2, 6, 7, 10, II, and 12 have a C and are not). As discussed later, the extent of this de-

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Figure 1. Analysis of Single and Double Point Mutants in the U6 Initiation Region

(A) Injection of U6 Initiation Region Mutants with U6 Maxigene. Wild-type U6 (lane 1) and a series of single and double point mutants in the initiation region were coinjected with a U6 maxigene into Xenopus oocyte nuclei along with [a-32P]GTP After incubation, transcripts were extracted and analyzed. The sequences of the mutants are displayed below the figure. The arrowheads above the G indicate the initiation nucleotide of wild-type U6. Dots indicate sequence identity with wild-type. The transcripts indicated are U6, U6 maxigene (U6M), and endogenous 5s. The mutant in lane 3 is altered upstream of -2 and behaves like wt. (B) Single injection of U6 initiation region mutants. Wild-type U6 and a series of single and double point mutations were analyzed by microinjection into Xenopus oocyte nuclei. The mutant sequences are below the figure. (C) Primer extension of wild-type and mutant U6 transcripts. RNA was extracted from control oocytes (lane C) and from oocytes microinjected into the nucleus with wild-type and various mutant U6 genes and analyzed by primer extension. The mutant sequences are below the figure. TCGT is the wild-type sequence, U6M the U6 maxigene. The lanes marked 2+5 were coinjected with the mutants used in lanes 2 and 5. a-amanitin was iniected as indicated. Maior POI II and Pol Ill start sites are marked II and Ill. P is the position of the primer. The mutant in lanes 3 and 10 is altered ~ upstream of -2 and behaves like wt.

pendence is unusual in a eukaryotic promoter. Secondly, examination of the transcripts arising from mutant genes that have a second purine inserted into the TCGT shows an apparent change in transcript length (compare lanes 4 and 5 with lane 3 and lane 9 with lane 8 in Figure 1A). Both of these efffects were analyzed in more detail.

The possibility that the TCCT mutant was not transcribed due to competition with the U6 maxigene was ex- amined. Single injection of the wild-type and +l G to C mutant, however, gave rise to a similar result (Figure lB, lanes 1 and 2). Also aTCTT mutant (Figure lB, lane 8) was only transcribed to roughly 10% of the wild-type level. We reasoned that these results could be explained in two ways. Either a transcription factor or RNA polymerase Ill itself was specifically interacting with the initiation region in a sequence dependent manner. If a transcription factor were involved, second-site mutations in the TCCT mutant would be unlikely to restore transcription, whereas if pol III were involved, second-site insertion of purines might restore activity due to the preference of pol III for purines as start sites (Sakonju et al., 1980). Our results favor the second possibility since the mutants fall into two classes. Those only containing pyrimidines at positions -2, -1, and +2 (all combinations with C at +l were tested) are poorly transcribed (Figure 16, lanes 2, 3, 5, and 8), while second-site changes to purines at -2, -1 (data not shown), or +2 in the context of the TCCT mutant resulted in a reversion to between 30% and 50% of wild-type transcription efficiency (Figure lB, lanes 4, 6, and 7). These results suggest that a reduction in the efficiency of interac-

tion between a purine-dependent factor, perhaps pal 111, and the initiation site of the U6 gene is responsible for the reduction in transcriptional efficiency of the TCCT and TCTT mutants. The fact that the TCCG and ACCT mutants are transcribed efficiently on single injection (Figure lB, lanes 4 and 6) but not when coinjected with the U6 maxigene (Figure lA, lanes 7 and 12) shows that nearby purines cannot fully compensate for the lack of a guanosine at +l and that templates with a C at +l have lost the ability to compete for a Pans-acting factor.

The U6 Promoter Has Dual Polymerase Specificity The reason for the apparent change in transcript length seen when additional purines were present in the initiation region mentioned above was next investigated. The U6 gene and various mutants were injected singly into oocytes and their transcripts analyzed by primer extension. Control oocytes at this exposure level give rise to a single primer extended band of 64 nucleotides (Figure lC, lane C) due to the endogenous U6, which is complementary to the primer. Injection of the wild-type U6 gene leads to an increase in this signal and to the appearance of severai new bands, the longest of which correspond to starts at the A and G residues at positions -6 and -5 (Figure lC, lane 1). The TCCT as well as the ACCT and TCGG mutants give rise to novel starts, the most prominent being at +3 (a G residue) in the TCCT and ACCT cases (Figure lC, lanes 2 and 4) and at the G inserted at +2 in the TCGG mutant (Figure lC, lane 5). Although some of these results are not easily explicable, why for example should a change

The RNA Polymerase Specificity of U snRNA Genes 437

B Figure 2. Analysis of Clustered Point Mutants

234567C in the U6 Promoter Region

1 (A) Injection of 116 promoter mutants with U6 maxigene. Analysis of total transcripts from oocytes microinjected with a U6 maxiqene and either wild-type U6 or promoter mutants created by site-directed clustered point mutation of 8 bp at a time. In lanes 2-11, the mutated bases are -73 to -80, -65 to -72, progressively to -1 to -8. The mutants defining the PSE and TATA regions are indicated. The clustered mutations always introduced the sequence AAGATCTG, which includes a Bgtll restriction site. (B) Primer extension ana@is of transcripts

ii 4 P

5 2 arising from a subset of the mutants analyzed in 2A. The mutants used are indicated below the figure In lane C, RNA from control oocytes was used. The major pot II and pot Ill start sites of wild-type U6 are indicated.

to A at -2 give rise to efficient use of the G at +3 as a start site (see Discussion), the most arresting feature was that the upstream starts were at exactly the same distance downstream of the U6 PSE homology as U2 starts are from the U2 PSE, i. e., 50 nucleotides (Skuzeski et al., 1984; Mattaj, 1986). Vve therefore tested whether they might be due to transcription by RNA polymerase II by coinjection with a-amanitin to a final concentration of 0.2 pglml. The results in Figure lC, lanes 7-13 show that while the transcripts initiating at +l, +2, and +3 in the various mutants are a-amanitin resistant and therefore pol III transcripts, the upstream starts are inhibited and therefore represent pal II transcripts. On longer exposure of similar gels, transcripts of the same length as the -6 and -5 starts are seen in control oocyte RNA (which contains stored endogenous U6 gene transcripts), but we are un- able to say whether these have arisen by RNA polymerase II transcription. The position of the 3’ ends of the U6 pol II transcripts from microinjected genes has been investigated by Sl mapping (data not shown). Among the many 3’ends detected, the most frequent corresponded to a position :oughly 80 bp downstream from the U6 coding region. No transcripts of this length that hybridized to a U6 probe were seen when oocyte RNA was analyzed on a Northern blot. It should also be noted that we do not detect pol II transcripts of defined length on microinjection of U6 (e.g., Figure 1A). Presumably, they are either too long or too diffuse to be detected.

The U6 Promoter Is Tripartite Having established that the initiation region was important, we wished to analyze U6 promoter structure further. To this end, a series of ten clustered point mutants was constructed in which 8 bp at a time between the positions -1 and -80 were converted to AAGATCTG (i.e., -80 to -73, -72 to -65, . ., -8 to -1). Their transcription was analyzed first by coinjection with the US maxigene. Two regions whose mutation results in complete loss of activity were seen. One corresponds to the PSE homology, which extends from -66 to -56 (Krol et al., 1987) and which is altered in the clones injected in Figure 2A, lanes 3,4, and 5. The second corresponds to the sequence GCTTAT- AA(G), the 3’ portion of which bears a resem pol II TATA signal (Breathnach and Chambon, 1981) which is altered in the clone injected in Figure 2A, iane 8. A sig- nificant drop in transcription is also seen when sequences between -9 and -16 are altered (Figure 2A, lane IO). As previously discussed, the U6 transcripts seen by analysis of total oocyte RNA are, at least mainly, pol fff transcripts. To analyze the effects of the clustered point mutations on U6 poi II transcription, we performed a primer extension analysis. Due to the high endogenous background, this assay is not suitable for accurate quantitation of pol III transcription of the mutants. However, the transcript accumulation results (Figure 2A) make this unnecessary. The -73 to -80 mutant (Figure 2A, iane 2) was used as a control. Comparing -73, -80 with the PSE mutants,

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amanitin . * . * ii3 456ii91011

Figure 3. Primer Extension Analysis of Spacing Mutants of the U6 Pro- moter.

C, control oocytes; wt, wild-type U6. The position of mutation and whether it was by insertion of 4 bp (+4) or deletion of 8 bp (-8) are indicated below the figure. a-amanitin was coinjected as indicated. The major pol II and pol Ill start sites on wild-type U6 are marked.

differential effects are seen. The -57, -64 and -49, -56 mutants are strong down mutations for both pol II and pol III transcription (Figure 2B, compare lanes 3 and 4 with lane 1 and with lane C, which represents the endogenous pol Ill background). This means that the PSE is essential for both pol II and pol Ill transcription of the U6 gene in Xenopus oocytes. The -65, -72 mutant does not appear to be strongly affected in this single injection experiment (Figure 28, lane 2) while it is not transcribed in the presence of the U6 maxigene (Figure 2A, lane 3). This sug- gests that the -65, -72 mutant can still bind weakly to the PSE transcription factor(s) but cannot compete with the maxigene efficiently for binding.

The TATA region mutant (-25, -32) shows strongly reduced accumulation of pol III transcripts (Figure 2A, lane 8) but apparently unaffected levels of pol II transcription (Figure 26, lane 5), suggesting that the pol II and pol III transcription units of the U6 promoter can be dissociated, both requiring the PSE but only pol Ill requiring the TATA and initiation site (see Figure lC, lane 2) elements. In other words the TATA mutant converts the U6 promoter into a pure pol II promoter indistinguishable from any Ul-U5 snRNA gene promoter in its basic properties.

The Roles of PSE and TATA in Start Site Selection The initiation site of pol II U snRNA genes is chosen by its distance downstream of the PSE (Skuzeski et al., 1984; Mattaj, 1986) while in other pol II transcription units, the TATA element fulfills this function (Grosschedl and Birn- stiel, 1980; Benoist and Chambon, 1981). We wished to determine what was responsible for start site selection In the two overlapping U6 promoters. To this end we made

four mutants. Jn two, 4 bp were inserted at either position 20 (between TATA and +I) or at position 36 (between PSE and TATA). In the other two, 8 bp were deleted from -12 to -19 (between TATA and +l) or from -36 to -43 (between PSE and TATA). Relative orientation of factor binding sites on the DNA helix is important for the activity of some prokaryotic and eukaryotic promoters (Takahashi et al., 1986; Dunn et al., 1984) and we expected these mutants to give us information on possible interactions of this type in the U6 promoter.

Analysis of U6 transcripts upon microinjection of these mutants showed that the 8 bp deletion between PSE and TATA very strongly reduced accumulation of transcripts while the other three mutants were all active in this assay (data not shown). A primer extension analysis of RNA from oocytes injected with these mutants in the presence and absence of 0.2 pgiml of a-amanitin is shown in Figure 3.

Several conclusions can be drawn. Neither of the 4 bp insertions affects the pol Ill start site (Figure 3, compare Banes l-3 with lanes 4-7), i.e., when 4 bp insertions are made between upstream promoter elements and the initiation site, the factor that interacts with the initiation site (which we have argued above may be pol Ill) determines where transcription starts. Pol III initiation however is altered in the 8 bp deletion mutants, No new pol Ill starts are detected when 8 bp are deleted between PSE and TATA (Figure 3, compare lanes 10 and 11 with lane 1). Together with the strong reduction in transcription observed when total oocyte transcripts are analyzed, this indicates that the pol Ill promoter cannot function when the PSE and TATA are too close together, perhaps because their cog- nate factors interfere with each other’s binding. When 8 bp are deleted between TATA and +l, new pol Ill starts are seen (Figure 3, lanes 8 and 9, some of the sample in lane 8 has been lost). These starts are mainly at cl2 rather than at +8. The sequence from +7 to +I2 is GCTTCG. Two things are noteworthy. First, the G at +7 is not used. This is analogous to our observations above that all new pol Ill starts seen in the U6 point mutants are downstream of +l (Figure 1C) and indicate that there is a block to starts upstream of a certain distance from the TATA (or PSE) element. Second, the sequence from -5 to +l in wild-type U6 is GTTTCG, remarkably similar to the sequence preceeding the G utilized in this mutant. The sequence of the U6 initiation region is discussed later.

The data on the pol II starts of these mutants (those starts present in lanes 4, 6, 8, and 10 but not in lanes 5, 7,9, and 11) indicate that two factors influence the start site choice. The first is distance from the PSE. All starts are close to 50 bp downstream from the PSE, independently of whether insertion or deletion has taken place between PSE and TATA or TATA and +l. The second factor is that there is an obvious preference for starts on purines roughly 50 bp from the PSE. In conclusion, the PSE directs the position of pol II initiation while pol Ill initiation sites are chosen by at least two interacting effects-a minimal distance between the start site and upstream elements and an apparent direct selection of the start site by an interacting factor or polymerase.

The RNA Poiymerase Specificity of U snRNA Denes 439

5 6 amanitin - -

Figure 4. Activity of Hybrid W/U6 Promoters

(A) Hybrid U2/U6 Promoters. A diagrammatic representation of the wild-type U6 gene (1) showing the DSE (enhancer), PSE, TATA, and initiation (+?) regions important for transcription. Parts 2 and 3 are different U2/U6 hybrid promoters that differ in whether or not they contain the U6 TATA element. The spacing between the PSE and +I is 56 bp in U6 and 55 bp in the two recombinant constructs. Not drawn to scale. (5) Transcription of the constructs shown in Figure 4A. The three constructs shown in Figure 4A were microinjected in the absence (lanes 1-3) and presence (lanes 4-6) of a-amanitin injected at 2 uglmt. (C) Primer extension of the constructs shown in Figure 4A. RNA was extracted from control oocytes (C) and from oocytes microinjected with the constructs shown in Figure 4A in the presence and absence of a-amanitin as indicated and analyzed by primer extension. Lanes 1 and 2, wild-type U6; lanes 3 and 4, construct 2; lanes 5 and 6, construct 3. The major pol II and pol III starts in the wild-type U6 promoter are indicated.

Conversion of the U2 Promoter to pol III Specificity The Xenopus U2 gene is, like other vertebrate Ui-U5 genes, transcribed by RNA polymerase II (Mattaj and Zeller, 1983). From the introductory discussion of U snRNA gene promoter structure and our results so far, it seemed possible that the U2 promoter could be converted to the use of pol Ill by the addition of the TATA and initiation elements of U6. In order to test this we made the constructs diagrammed in Figure 4A. Part 1 is the U6 gene, and parts 2 and 3 are recombinants between two of the U6 clustered point mutants and two U2 mutants previously described, which had BamHl sites inserted into con- venient positions of the U2 promoter (see Experimental Procedures and Mattaj, 1986). Relative to U6, the distance between +l and the PSE is shortened by 1 bp in both constructs. Since it was known that the U6 coding region downstream of +l is not necessary for promoter function or efficiency (Das et al., 1988) and since our data (Figure 2) show that even when the different elements of the U6 promoter are mutated this does not unmask the activity of possible redundant signals, we concluded that the coding region is transcriptionally inert and we have used it as a reporter transcript in constructs 2 and 3.

The transcriptional activity of these constructs is shown

in Figure 46. Lanes l-3 in Figure 4 show the activity of the U6 gene and constructs 2 and 3 in the absence and lanes 4-6 (Figure 4B) in the presence of 6.2 Kg/ml of a-amanitin. Clearly, construct 3 in which the TATA and +l regions are both present behaves very much like wild-type U6, while construct 2, even on this overexposure, does not produce detectable pol III transcripts. Analysis of these constructs by primer extension revealed a surprise. While wild-type U6 and construct 2 produced poi II transcripts as expected (Figure 4C compare lanes I and 2,3 and 4), construct 3 is apparently only active in pot II1 transcription (Figure 4C, compare lanes 5 and 6). Although the lack of pal II transcription is unexpected the transcription of this construct by pol Ill shows that it has been possible to change the polymerase specificity of the U2 promoter.

Discussion

The Xenopus U6 promoter consists of two parts: a coliec- tion of elements similar to the PSE and DSE of pol II snRNA gene promoters that enable its transcription by RNA polymerase II, and two additional sequences, which, in combination with the pol II elements, are required for transcription of the gene by RNA po~yme~ase 311.

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One other gene, c-myc, has been shown to have both pal II and pol III specificity (Chung et al., 1987) although on the basis of the extreme template concentration dependence of the pol Ill activity in oocyte microinjection experiments and the lack of evidence for pol Ill transcription of c-myc in vivo it has been suggested that this may be artefactual (Bentley and Groudine, 1988). The evidence that pol Ill transcription of U6 is genuine was reviewed above, but the likelihood that the a-amanitin-sensitive transcription from the U6 promoter by pol II is artefactual should be considered. The absolute dependence on the PSE of U6 pol II transcription is reminiscent of other pol II U snRNA promoters, as is the dependence on the distance between the PSE and the initiation site. The U6 DSE or enhancer (Bark et al., 1987; Carbon et al., 1987; Das et al., 1988; Kunkel and Pederson, 1988) is extremely similar to pol II U snRNA gene DSEs. In fact, all the elements of U6 and U2 promoters are interchangable (Bark et al., 1987; Carbon et al., 1987; Das et al., 1988; Kunkel and Peder- son, 1988; this paper and H. D. Parry, G. Tebb, and I. W. Mattaj, unpublished data). In other words, by most if not all of the criteria by which any of the other U snRNA genes is a bona fide pol II gene U6 is also.

The extreme dependence of the U6 promoter on sequences at and immediately adjacent to the initiation site is unusual. Alteration of the guanosine nucleotide at +l to a pyrimidine resulted in a drop of at least 20-fold in U6 transcription by pol Ill (Figure 1), while alteration to an ade- nine resulted in a decrease of roughly 3-fold (data not shown). In similar studies of other higher eukaryotic pol II and pol Ill genes, deletion of the initiation region resulted in a minor effect on promoter efficiency (Sakonju et al., 1980; Grosschedl and Birnstiel, 1980; Benoist and Cham- bon, 1981). In such mutants, transcription initiated at a position dictated by distance from either gene external (Grosschedl and Birnstiel, 1980; Benoist and Chambon, 1981) or internal (Sakonju et al., 1980) promoter elements. Mutants in which the spacing between different elements of the U6 promoter were changed (Figure 3) indicate that at least two factors affect the choice of the pol Ill initiation site. A minimal distance must be preserved between the start site and (an) upstream promoter element(s), and there is apparently direct selection of the start site by an interacting factor or polymerase. Mutants in which +1 is changed to a pyrimidine lose the ability to bind strongly to this factor (Figure 1).

To our knowledge, the only other eukaryotic examples of sequence dependence at the initiation site of nuclear promoters occur in yeast. Studies of several yeast promoters (Chen and Struhl, 1985; Nagawa and Fink, 1985; Hahn et al., 1985; Rudolph and Hinnen 1987) led to the conclusion that yeast RNA polymerase II initiates prefer- entially at certain sequences. From sequence analysis, two consensus start sites were suggested, TC(Pu)A and PyPyPuPyPy, where the Purine residue is at +l in each case (Hahn et al., 1985; Rudolph and Hinnen, 1987). From the analysis of starts in the various U6 mutants, we can suggest that PyPyPyG is the preferred initiation sequence for the U6 gene. If this is correct, it explains some of the puzzling data obtained by point mutation of the initiation

region where, for example, insertion of an A at -2 caused new starts at the G at +3 (Figure IC). This G is part of a PyPyPyG stretch in the mutant. In comparing the yeast and U6 results, it is important however to remember that the initiation site is only necessary for accuracy in yeast while it is required for promoter efficiency in U6. Alteration of the sequence at a yeast initiation site leads to the efficient choice of other consensus start sites nearby.

The similarity of one of the elements of the U6 promoter to the pol II TATA box consensus (Breathnach and Cham- bon, 1981) raises the question of whether the same factor recognizes both of these elements. This problem is com- plicated by the recent observation that pol II TATA binding factors fall into at least two functional classes (Simon et al., 1988). We cannot provide a definitive answer but favor the possibility that the factors are different for the following reasons. First, the TATA in U6 is a pol Ill determinant. There is nothing in its position to indicate why it would not act in the transcription of this gene by pol II but it does no!. Second, we previously made and analyzed a mutant that converted the CTTATAAG sequence to TTTAAAAG. The mutation had no marked effect on transcription, whereas analogous mutants in pol II TATA boxes have an inactivat- ing effect (Carbon et al., 1987). Third, several pol Ill genes have functionally important upstream “TATA boxes” (e.g., Murphy et al., 1987; Morton and Sprague, 1984; Tyler, 1987). Their sequences, and that of the U6 point mutation mentioned above, do not always closely resemble the sequences of pol II TATA boxes of various classes (Simon et al., 1988).

Two possible (not mutually exclusive) mechanistic roles for the PSE in the activation of pol Ill transcription can be imagined. Either it might directly interact with other factors involved in transcription initiation in conjunction with the TATA factor, or it might act as an adaptor to channel upstream activation signals to the TATA factor. Our mutational results and those of Kunkel and Pederson (1988) with human U6 favor the former idea since PSE mutants are totally inactive both in the oocyte and in transfected 293 cells. Results obtained by in vitro transcription of the human 7SK gene (Murphy et al., 1987) whose promoter is extremely similar to the human U6 gene promoter (Kunkel and Pederson, 1988), show that while a TATA mutant is totally inactive, deletion of the 7SK PSE leaves a basal level of activity. This result might favor an “adaptor” model. In vitro reconstitution experiments with purified factors may help to answer this question.

It has previously been suggested (Murphy et al., 1987) that genes of the 7SKIU6 type closely resemble prokaryotic genes and thus may be very ancient. If RNA polymerase Ill does interact directlywith the initiation region of U6, this would strengthen the analogy with prokaryotic promoters. Our results have, in any case, relevance for promoter evolution since they demonstrate that relatively simple mutational events can change the RNA polymerase utilized by a promoter. Different eukaryotic and prokaryotic polymerases probably derived from a common ancestor (Allison et al., 1985; Biggs et al., 1985). Multiple polymerases in eukaryotes, having arisen by gene duplication, would have had the potential to coevolve with different

The RNA Polymerase Specificity of U snRNA Genes 441

promoters and transcription factors to produce the diver- sity of transcriptionat units now present. Genes like U6 may have retained the ability to utilize different combinations of factors and polymerases either for regulatory reasons, in order to produce transcripts of different structure, or simply because the utilization of different polymerases was of no evolutionary disadvantage to the gene.

Experimental Procedures

General Methods Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and DNA polymerase Klenow fragment were obtained from Boehringer Mannheim and used following the procedures of Maniatis et al. (1982). Cloning into Ml3 vectors and preparation of double-stranded and single-stranded DNA were as described (Messing, 1983). DNA sequence anaiyses were carried out using the dideoxy chain termination method (Sanger et al., 1977) with [a-35S]dATP (Amersham) as the labei.

Mutagenesis and Promoter Constructs Oiigonucleotides were synthesized with the phosphoramidite method (Sinha et al., 1984) on an Applied Biosystems synthesizer and used for mutagenesis by the method of Taylor et al. (1985). The +4 spacing mutants were made by digesting the -9, -16 and -33, -40 clustered point mutants with Bglll, filling in the overhanging tails with the Klenow fragment of DNA polymerase I, and re-ligating. The -8 mutants were made by rejoining promoter fragments digested with Bglll and BamHl from two pairs of clustered point mutants. All mutants were checked by DNA sequencing. Manufacturers’ recommendations for enzyme reaction conditions were followed.

The U2/U6 hybrid promoters were created by joining the promoter fragments of the U2 mutants P48, 4 and P23, 4 (Mattaj, 1986) to the coding region fragments of the U6 clustered point mutants -49, -56 and -9, -16, respectively. This was acheived by digesting the U2 mutants with BamHl and the U6 mutants with Bglll and BamHl and insert- ing the correct U6 fragments into the U2 constructs by ligation. The orientation of insertion was determined by restriction enzyme analysis.

Oocyte Microinjection Oocytes were microinjected, aiming at the nucleus, with 30-50 nl of DNA at a concentration of 250 nglml. When a-amanitin was used, it was added to the DNA solution to a concentration of 2 pglml. The final Concentration in the oocyte is 5%-10% of this value. [ I@S]GTP (Amersham; 400 Cilmmol, 0.1-0.5 nCi/oocyte) was coinjected with the DNA. RNA was extracted and analyzed as described (Mattaj and De Robertis, 1985). Oocytes were analyzed in batches of ten. Half an oocyte equivalent (approximately 2.5 pg) of RNA was loaded onto the gel.

primer Extension The oligonucleotide primer used had the sequence 5’-TCATCCTTG- CGCAGGGGTCTCTGTATCGTT-3’. The extended cDNA product of wild-type U6 RNA transcribed by RNA polymerase IO had a length of 64 nucleotides. The procedures used for hybridization and cDNA synthesis were from Bensi et al. (1985).

Acknowledgments

We wish to thank Graham Tebb, Jorg Hamm, Angus Lamond, Lennart Philipson, Aiccardo Cortese, Paolo Monaci, and Alfred0 Nicosia for commenting on the manuscript, Jean-Pierre Ebel for his support and encouragement, Annie Hoeft for oligonucleotide synthesis, Petra Riedinger for drawing the figures, and Heide Seifert for typing the manuscript.

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

Received June 27, 1988; revised August 5, 1988.

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changing the rna polymerase specificity of u snrna gene promoters

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