snrna genes: a model system to study fundamental mechanisms of transcription … · 2001-06-04 ·...
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snRNA genes: a model system to study fundamental mechanisms of transcription
Nouria Hernandez
Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
11724.
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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on June 4, 2001 as Manuscript R100032200 by guest on M
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The human small nuclear RNA (snRNA) genes, which encode snRNAs that are involved in RNA
processing reactions such as mRNA splicing, serve as prototypes for a family of genes whose
promoters are characterized by the presence of a proximal sequence element (PSE) and a distal
sequence element (DSE). From a transcription point of view, this family of genes is highly
interesting because all its members have very similar promoters, even though some of them are
transcribed by RNA polymerase (pol) II and others by pol III. As a result, the snRNA genes have
served as a model system to explore how RNA polymerase specificity is determined, and, in
general, to compare the pol II and III transcription machineries. This has led to the concept that
the pol II and III transcription machineries use common factors, the best known of which is the
TATA box binding protein TBP. In addition, the relative simplicity of these promoters has also
made them an attractive system to study how transcriptional activators perform their function.
The structure of snRNA promoters.
Figure 1 shows the structures of snRNA promoters from Homo sapiens (Hs), Arabidopsis
thaliana (At), and Drosophila melanogaster (Dm), and serves to illustrate the remarkable fact that
although snRNA promoters have diverged during evolution, the close similarity between those
recognized by pol II and those recognized by pol III has been conserved. In fact, in each of the
examples in Figure 1, RNA polymerase specificity can be changed by altering a single
parameter, indicated in red in the figure.
In the human genes, the U1 and U2 snRNA promoters serve as the prototypic pol II snRNA
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promoters, and the U6 snRNA promoter serves as the prototypic pol III snRNA promoter (see (1)
for a review). The human pol II snRNA core promoters contain only one essential element, the
proximal sequence element (PSE), whereas the pol III snRNA core promoters consist of two
elements, the PSE and a TATA box located at a fixed distance downstream. The distal sequence
element (DSE) serves to enhance transcription from the core promoter. Both the DSE and the
PSE can be interchanged between pol II and III snRNA promoters with no effect on RNA
polymerase specificity, which is determined by the presence or absence of the TATA box. The
A. thaliana pol II and III snRNA promoters contain an upstream sequence element (USE) and a
TATA box, which are both interchangeable between the pol II and III snRNA promoters. RNA
polymerase specificity is determined in this case by the exact spacing between the USE and the
TATA box, which is 33 to 34 base pairs (bp) and 23 to 24 bp in the pol II and III snRNA
promoters, respectively (2).
The D. melanogaster pol II snRNA promoters contain two elements referred to as the PSEA and
the PSEB spaced by eight bp, and the pol III snRNA promoters contain a PSEA and a TATA box
spaced by 12 bp. The PSEA is quite conserved in various pol II and III snRNA promoters but
positions 19 and 20 of the 21 bp elements are always g/aG in the pol II, and TC in the pol III,
snRNA promoters. RNA polymerase specificity is determined by the precise sequence of the
PSEA element, with the base pairs at positions 19 and 20 playing a major role ((3), and
references therein).
A number of snRNA promoters have been characterized in various sea urchins. Like in other
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species, the pol II and III snRNA promoters are closely related in structure. They all have a PSE
and some have a TATA box, but the presence of the TATA box does not correlate with RNA
polymerase specificity. The PSEs in different snRNA promoters show little sequence identity
and yet can be exchanged with no effect on polymerase specificity. The determinants of RNA
polymerase specificity are not known ((4), and references therein). In Saccharomyces cerevisiae,
only the pol III U6 snRNA promoter has been studied. It consists of a TATA box located
upstream of the transcription start site, and A and B boxes typical of gene-internal tRNA
promoters. The A box is located, as in tRNA genes, within the RNA coding region, but the B
box is located at an anomalous position 3’ of the gene (5-7).
The PSE binding factors.
The snRNA promoters in vertebrates, A. thaliana, D. melanogaster, and sea urchins all contain an
element, variously called the PSE, PSEA, or USE, centered 50 to 70 bp upstream of the
transcription start site. The factor binding to this element has been best characterized in the
human system, and is variously known as PBP, PTF, or SNAPc. It is a complex containing five
types of subunits, SNAP190, SNAP50 (PTFβ), SNAP45 (PTFδ), SNAP43 (PTFγ), and SNAP19
(see (8) and references within). SNAP190 forms the backbone of the complex, with SNAP19
and SNAP45 associating toward the N- and C-terminus, respectively, of the molecule. SNAP43
can associate with the same region of SNAP190 as SNAP19, and SNAP50 joins the complex by
associating with SNAP43 (see Figure 3 below for an illustration SNAPc).
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The ability to assemble recombinant SNAPc and, thus mutant forms of SNAPc has allowed a
study of the role of SNAPc subunits for binding to DNA, and for basal and activated
transcription by RNA polymerases II and III. The smallest subassembly of SNAPc subunits
tested that binds to the PSE with the same specificity as the complete complex consists of
SNAP190 aa 84-505, SNAP43 aa 1-268, and SNAP50 (9). This observation is consistent with
UV crosslinking experiments that suggest that within SNAPc, both SNAP190 and SNAP50 are
in close contact with the DNA (see (8) and references therein). The specific binding of SNAPc
to the PSE is mediated in part by an unusual Myb domain extending from aa 263 to 503 within
SNAP190 and containing a half repeat followed by four repeats (8,10).
In the human system, the very same SNAPc is involved in transcription by pol II and pol III (11).
The polypeptide composition of PSE-binding factors from other species is unknown, but there
are indications that at least in some cases, the same PSE binding factor is also recruited to both
pol II and III snRNA promoters. Thus, both in sea urchins and D. melanogaster, similar
complexes bind to the PSEs of pol II and III snRNA promoters as judged from electrophoretic
mobility shift assays (EMSAs) (3,4), and in the latter case, site-specific protein-DNA photo
cross-linking experiments reveal the same set of polypeptides in close proximity to the DNA in
both cases. Interestingly, however, the precise crosslinking patterns of these polypeptides to the
U1 and U6 PSEAs are significantly different. Thus, in D. melanogaster, RNA polymerase
specificity may ultimately be determined by different conformations of the same factor, which
are dictated by the exact PSEA sequence (12).
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Factors besides SNAPc required for pol II transcription of snRNA genes.
Transcription from TATA-box containing mRNA promoters can be reconstituted with a
combination of recombinant and well-defined factors, as shown in Figure 2A. In vitro, these
factors can be added sequentially to the promoter to form a functional transcription initiation
complex, and each step can be monitored by EMSA. TBP or the TBP-containing complex
TFIID binds first to the TATA box, followed by TFIIB, a TFIIF/Pol II complex, TFIIE, and
TFIIH. TFIIA can join the initiation complex at any stage of assembly, and its main role for
mRNA core promoter function appears to be counteracting repressors that associate with TBP
and prevent its binding to DNA (13).
In the case of the pol II snRNA promoters, the transcription initiation complex has not yet been
assembled in a stepwise fashion in vitro, but many of its components have been identified
functionally by depletion of transcription extracts with specific antibodies and reconstitution of
transcription with recombinant factors. Thus, many of the players are known but their mode of
assembly on snRNA promoters is not, and thus their location in Figure 2A is arbitrary.
Depletion and reconstitution experiments indicate that recombinant TBP (but not the TBP-
containing complexes TFIID or TFIIIB), TFIIB, TFIIA, TFIIF, and TFIIE are required ((14), and
references therein). TFIIA appears to perform a more direct function in snRNA transcription
complex assembly than just counteracting TBP-associated repressors. The role, if any, of TFIIH
in pol II transcription of snRNA genes is not clear. Depletion and reconstitution experiments
suggest that U1 transcription either does not require TFIIH, or requires much lower levels than
transcription from a mRNA promoter. If TFIIH is indeed not required, this raises the interesting
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question of how open complex formation is achieved at snRNA promoters. A combination of all
the general transcription factors and SNAPc does not initiate transcription from the U1 promoter,
suggesting that additional as yet unidentified factors are required (14).
Factors besides SNAPc required for pol III transcription of snRNA genes.
The key player for recruitment of pol II to a promoter is the factor TFIIB, because it contacts the
RNA polymerase directly. In the case of pol III, this role is played mainly by the multisubunit
factor TFIIIB. TFIIIB was completely defined first in S. cerevisiae and consists of three
subunits, TBP, a tightly associated subunit referred to as the TFIIB-related factor BRF1
(PCF4/TDS4) (see (15) for references), and a more loosely associated polypeptide called B"
(TFIIIB90/TFC5/TFC7) (16,17). This TFIIIB complex is involved in transcription from all types
of yeast pol III promoters tested including the gene-internal tRNA-type promoters and the U6
promoter, which, as described above, contains a TATA box and A and B boxes (18).
TBP was shown to be required for pol III transcription of vertebrate snRNA genes before TBP
was known to be a subunit of TFIIIB (see (15) for a review). Ironically, however, the
composition of mammalian TFIIIB and the role of TFIIIB polypeptides other than TBP in
snRNA gene transcription have been determined only recently. A human homologue of yeast B"
was recently cloned (19). The protein shows strong similarity to the yeast protein within and
around a 59 aa domain called the SANT domain, which is essential for transcription in yeast.
Depletion of human B" (hB") from transcription extracts debilitates transcription from the U6
promoter and a tRNA-type promoter, and transcription can be restored in both cases by addition
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of recombinant hB" (19). hB" is, therefore, shown as part of the initiation complex assembled on
both U6 and tRNA-type promoters in Figure 2B.
The first human homologue of yeast BRF cloned was called TFIIIB90 (20) or human BRF
(hBRF) (21). Like its yeast counterpart, and like its cousin TFIIB, the protein has a zinc binding
domain at its N-terminus followed by a core domain consisting of two degenerate repeats. The
C-terminal half of the protein is poorly conserved with the yeast protein, and has no counterpart
in TFIIB. Depletion and reconstitution experiments have shown that human BRF is required for
transcription from tRNA-type promoters, but not for transcription from the U6 snRNA promoter
(21). Remarkably, as shown in Figure 2B, the U6 snRNA promoter uses another homologue of
yeast BRF, human BRFU (19), also called TFIIIB50 (22). hBRFU represents another member of
the TFIIB-related family of proteins and has conserved zinc and core domains, and a divergent
C-terminal domain.
Human BRFU was cloned both through database searching of proteins similar to hBRF (19) and
through purification of a complex consisting of BRFU and four tightly associated polypeptides
(22). The role of the BFRU-associated polypeptides is presently not clear. In one series of
experiments, U6 transcription could be restored in a BRFU-depleted extract by addition of
recombinant BRFU expressed in E. coli (19), and a combination of partially purified pol III and
recombinant SNAPc, TBP, hB", and hBRFU could direct efficient U6 transcription (23). In
another series of experiments, depletion of BRFU (TFIIIB50) debilitated transcription from a
snRNA-type promoter, but transcription could not be reconstituted by addition of recombinant
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BRFU. Instead, transcription could only be reconstituted by addition of a BRFU-containing
complex immuno-purified from cells expressing tagged BRFU (22). Thus, it is not clear
whether the BRFU-associated polypeptides in the BRFU-containing complex are essential for
U6 transcription.
BRF2, another factor encoded by an alternatively spliced BRF pre-mRNA, may also be required
for U6 transcription (24). BRF2 lacks the zinc finger domain and the first repeat that are present
in BRF and conserved in the other proteins of the TFIIB family, as well as the C-terminal
domain present in BRF. Depletion of extracts with antibodies recognizing all BRF variants
debilitated U6 transcription, and transcription could be specifically reconstituted by addition of
material immuno-purified from cells expressing tagged BRF2 (24). These results raise the
possibility that the U6 transcription complex contains two proteins related to BRF, BRFU and
BRF2.
The discovery of BRFU is an important step toward a complete understanding of how RNA
polymerase specificity is determined at the human snRNA promoters. Indeed, in mRNA
promoters, TFIIB associates with TBP bound to the TATA box and, in a manner absolutely
dependent on the zinc ribbon domain, with the pol II/TFIIF complex (13). Thus, at least for
mRNA promoters, TFIIB can be viewed as the key factor that bridges DNA-associated TBP or
TFIID with the polymerase, and it seems likely that TFIIB performs the same role in the pol II
snRNA promoters. Like TFIIB, BRF also contacts directly the RNA polymerase, in this case pol
III. The precise BRF domain required for this function is not known but it is not the zinc ribbon
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domain, because deletion or mutation of the zinc ribbon does not affect RNA polymerase
recruitment but instead affects open complex formation (see (19) for references). It seems likely
that in the human U6 promoter, the recruitment of pol III is accomplished by BRFU, either
through the zinc domain as for TFIIB and pol II, or through another part of the protein as for
BRF and pol III at tRNA-type promoters. Thus, the determination of RNA polymerase
specificity may ultimately depend on whether TFIIB or BRFU is recruited to the promoter.
Activation of snRNA gene transcription.
The human snRNA promoters are activated by a DSE. The DSE is composed of various protein
binding sites, but one of them is almost invariably the octamer sequence ATGCAAAT. In
addition, it has become clear that the DSEs of many snRNA genes contain an element referred to
as the SPH element. The SPH (for “Sph1 postoctamer homology”) element was first identified
in the chicken snRNA gene enhancer ((25,26), and references therein), and in the enhancer of the
selenocysteine tRNA gene, whose promoter contains a PSE and TATA box (27,28). It then
became clear that a functionally important element located immediately upstream of the octamer
sequence in the human U6 snRNA promoter called the NONOCT element (29) corresponds, in
fact, to an SPH element (30), and that SPH elements are present in the enhancers of many
snRNA promoters (31). In the human U6 snRNA promoter, both the octamer and SPH elements
stimulate the formation of preinitiation complexes (32).
The SPH motif recruits in vitro a transcription factor called Staf or SPH binding factor (SBF),
which was cloned first from Xenopus (31,33), and then from mouse (34) and humans (35,36).
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Xenopus Staf is a zinc finger protein containing seven zinc fingers of the C2-H2 type, different
sets of which can be used to bind to different DNA targets. In particular, zinc finger 1 is required
for binding to the selenocysteine tRNA enhancer but not to the U6 enhancer, where introduction
of a zinc finger 1 binding site interferes with binding of Oct-1, the protein recruited to the
adjacent downstream octamer sequence ((37) and references therein). Xenopus Staf contains two
separable activation domains capable of selectively stimulating transcription from snRNA-type
and mRNA-type promoters (38). The human proteins ZNF143 and, to a lesser extent, ZNF76,
are similar to Xenopus Staf, share similar DNA binding specificities, and can activate pol II and
III snRNA gene transcription (35,36).
The octamer sequence recruits the transcription activator Oct-1, as suggested by i) the broad
expression of Oct-1, which parallels the broad expression of snRNA genes; ii) the presence of
snRNA-specific transcription activation domains within Oct-1; and iii) the localization of Oct-1
to snRNA promoter sequences in vivo by chromatin immunoprecipitation experiments ((39), and
references therein; (40)). Oct-1 activates snRNA gene transcription not only through its
activation domains, but also through its POU domain, a bipartite DNA binding domain
consisting of two helix-turn-helix-containing DNA-binding structures: an amino-terminal
POU-specific (POUS) domain and a carboxy-terminal POU-homeo (POUH) domain, joined by
a flexible linker (41). As described further below, this results from the ability of the Oct-1 POU
domain to bind cooperatively with SNAPc and thus recruit SNAPc to the PSE.
Assembly of a stable snRNA transcription initiation complex.
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The characterization of many of the factors that bind to snRNA promoters has allowed a study of
how these factors interact with each other to form a stable transcription initiation complex. Our
current understanding of this process is summarized in Figure 3. Both TBP and SNAPc have
built in mechanisms that prevent their efficient binding to DNA on their own. In the case of
TBP, this “damper” of DNA binding resides in the N-terminus of the protein, because deletion
of this segment greatly increases the ability of the truncated protein to bind to TATA boxes (42).
Perhaps the N-terminus of TBP masks the DNA binding domain of the protein, as illustrated in
Figure 3A, although other scenarios are equally possible. In the case of SNAPc, the damper of
DNA binding resides somewhere within the C-terminal two thirds of SNAP190 and/or SNAP45,
because a mini-SNAPc lacking these sequences binds much more efficiently to DNA than
complete SNAPc (10). Both TBP and SNAPc dissociate slowly from the DNA and bind with
relatively low sequence specificity, so these built-in dampers may serve to ensure that these
factors not bind to inappropriate sites in the genome.
Figure 3B illustrates SNAPc, TBP, and the Oct-1 POU domain bound to DNA. The Oct-1 POU
domain and SNAPc bind cooperatively to their respective DNA binding sites, and so do SNAPc
and TBP (see (8) and references within). Very strikingly, the same regions of SNAPc and TBP
that serve as dampers of DNA binding are required for cooperative binding. Thus, the N-
terminal domain of TBP is absolutely required for cooperative binding with SNAPc, perhaps
because as illustrated in Figure 3B, it is engaged in a protein-protein interaction with SNAPc
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(42). Similarly, the C-terminal domain of SNAP190 is required for cooperative binding with
Oct-1, and in this case it is clear that cooperative binding is dependent on a direct protein-
protein contact between the two proteins, which involves a glutamic acid at position 7 within the
POUS domain (blue triangle in Figure 3B) and a lysine at position 900 within SNAP190 (Oct-1
interacting region “OIR” in Figure 3B) (43). Thus, cooperative binding of these factors probably
involves conformational changes that convert the dampers of DNA binding into handles that
contact and stabilize the factor binding to a neighboring site. This intricate mode of DNA
binding ensures, in effect, that factors bind to sites located in promoter sequences rather than to
inappropriate isolated sites.
The cooperative binding of the Oct-1 POU domain and SNAPc was originally characterized on
probes containing closely spaced octamer and PSE. In the natural snRNA promoters, however,
the octamer sequence and the PSE are separated by about 150 bp, and this distance prevents
cooperative binding of Oct-1 and SNAPc on naked DNA probes. However, mapping of DNase
I and micrococcal nuclease cleavage sites in chromatin suggests the presence of a positioned
nucleosome between the octamer sequence and the PSE in both the U1 and U6 snRNA
promoters (40,44). Indeed, in vitro chromatin assembly results in the positioning of a
nucleosome at the same location (40,45). Importantly, chromatin assembly allows the Oct-1
POU domain to activate transcription from the natural U6 promoter. It also allows cooperative
binding of the Oct-1 POU domain and SNAPc, and this cooperative binding is dependent on the
same direct protein-protein contact as cooperative binding to closely spaced sites on naked DNA
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(40). These results suggest that the role of the positioned nucleosome is to bring into close
proximity the octamer sequence and the PSE such that SNAPc and the Oct-1 POU domain can
contact, and recruit, each other to the DNA, as illustrated in Figure 3B. Thus, this is a case
where a nucleosome does not repress transcription but instead is a functional component of the
transcription activation process.
ACKNOWLEDGMENTS
I thank Winship Herr for comments on the manuscript. Our work on snRNA gene transcription
is funded in part by NIH grant GM38810. N.H. is supported by the Howard Hughes Medical
Institute.
REFERENCES
1. Lobo, S. M., and Hernandez, N. (1994) in Transcription, mechanisms and regulation.
(Conaway, R. C., and Conaway, J. W., eds), pp. 127-159, Raven Press, Ltd., New York
2. Waibel, F., and Filipowicz, W. (1990) Nature 346, 199-202
3. Jensen, R. C., Wang, Y., Hardin, S. B., and Stumph, W. E. (1998) Nucleic Acids Res
26(2), 616-22.
4. Li, J. M., Haberman, R. P., and Marzluff, W. F. (1996) Mol Cell Biol 16(3), 1275-81.
14
by guest on May 29, 2020
http://ww
w.jbc.org/
Dow
nloaded from
5. Brow, D. A., and Guthrie, C. (1990) Genes & Dev. 4, 1345-1356
6. Burnol, A. F., Margottin, F., Schultz, P., Marsolier, M. C., Oudet, P., and Sentenac, A.
(1993) J. Mol. Biol. 233, 644-658
7. Eschenlauer, J. B., Kaiser, M. W., Gerlach, V. L., and Brow, D. A. (1993) Mol. Cell.
Biol. 13, 3015-3026
8. Henry, R. W., Ford, E., Mital, R., Mittal, V., and Hernandez, N. (1998) in Cold Spring
Harbor Symposia on Quantitative Biology Vol. 63, pp. 111-120, Cold Spring Harbor
Laboratory Press
9. Ma, B., and Hernandez, N. (2000) J Biol Chem 30, 30
10. Mittal, V., Ma, B., and N., H. (1999) Genes Dev. 13, 1807-1821
11. Henry, R. W., Mittal, V., Ma, B., Kobayashi, R., and Hernandez, N. (1998) Genes &
Dev. 12, 2664-2672
12. Wang, Y., and Stumph, W. E. (1998) Mol. Cell. Biol. 18, 1570-1579
13. Orphanides, G., Lagrange, T., and Reinberg, D. (1996) Genes & Dev. 10, 2657-2683
14. Kuhlman, T. C., Cho, H., Reinberg, D., and Hernandez, N. (1999) Mol. Cell. Biol. 19,
2130-2141
15. Hernandez, N. (1993) Genes Dev. 7, 1291-1308
16. Kassavetis, G. A., Nguyen, S. T., Kobayashi, R., Kumar, A., Geiduschek, E. P., and
Pisano, M. (1995) Proc. Natl. Acad. Sci. USA 92, 9786-9790
17. Rüth, J., Conesa, C., Dieci, G., Lefebvre, O., Dusterhoft, A., Ottonello, S., and Sentenac,
A. (1996) EMBO j. 15, 1941-1949
18. Joazeiro, C. A., Kassavetis, G. A., and Geiduschek, E. P. (1994) Mol. Cell. Biol. 14,
15
by guest on May 29, 2020
http://ww
w.jbc.org/
Dow
nloaded from
2798-2808
19. Schramm, L., Pendergrast, P. S., Sun, Y., and Hernandez, N. (2000) Genes & Dev. 14,
2650-2663
20. Wang, Z., and Roeder, R. G. (1995) Proc. Natl. Acad. Sci. USA 92, 7026-7030
21. Mital, R., Kobayashi, R., and Hernandez, N. (1996) Mol. Cell. Biol. 16, 7031-7042
22. Teichmann, M., Wang, Z., and Roeder, R. G. (2000) Proc Natl Acad Sci U S A 97(26),
14200-14205
23. Sepehri Chong, S., Hu, P., and Hernandez, N. (2001) J. Biol. Chem., In press.
24. McCulloch, V., Hardin, P., Peng, W., Ruppert, J. M., and Lobo-Ruppert, S. M. (2000)
Embo J 19(15), 4134-43
25. Roebuck, K. A., Szeto, D. P., Green, K. P., Fan, Q. N., and Stumph, W. E. (1990) Mol
Cell Biol 10(1), 341-52.
26. Zamrod, Z., and Stumph, W. E. (1990) Nucleic Acids Res 18(24), 7323-30.
27. Myslinski, E., Krol, A., and Carbon, P. (1992) Nucleic Acids Res 20(2), 203-9.
28. Myslinski, E., Schuster, C., Krol, A., and Carbon, P. (1993) J Mol Biol 234(2), 311-8.
29. Danzeiser, D. A., Urso, O., and Kunkel, G. R. (1993) Mol Cell Biol 13(8), 4670-8.
30. Kunkel, G. R., Cheung, T. C., Miyake, J. H., Urso, O., McNamara-Schroeder, K. J., and
Stumph, W. E. (1996) Gene Expr. 6, 59-72
31. Schaub, M., Myslinski, E., Schuster, C., Krol, A., and Carbon, P. (1997) EMBO J. 16,
173-181
32. Kunkel, G. R., and Hixson, J. D. (1998) Nucleic Acids Res. 26, 1536-1543
33. Schuster, C., Myslinski, E., Krol, A., and Carbon, P. (1995) EMBO J. 14, 3777-3787
16
by guest on May 29, 2020
http://ww
w.jbc.org/
Dow
nloaded from
34. Adachi, K., Saito, H., Tanaka, T., and Oka, T. (1998) J Biol Chem 273(15), 8598-606.
35. Myslinski, E., Krol, A., and Carbon, P. (1998) J. Biol. Chem. 273, 1998-2006
36. Rincon, J. C., Engler, S. K., Hargrove, B. W., and Kunkel, G. R. (1998) Nucleic Acids
Res. 26, 4846-4852
37. Schaub, M., Myslinski, E., Krol, A., and Carbon, P. (1999) J Biol Chem 274(35), 25042-
50.
38. Schuster, C., Krol, A., and Carbon, P. (1998) Mol Cell Biol 18(5), 2650-8.
39. Das, G., Hinkley, C. S., and Herr, W. (1995) Nature 374(6523), 657-660
40. Zhao, X., Pendergrast, P. S., and Hernandez, N. (2001) Mol. Cell 7, 539-549
41. Herr, W., and Cleary, M. A. (1995) Genes & Dev. 9, 1679-1693
42. Mittal, V., and Hernandez, N. (1997) Science 275, 1136-1140
43. Ford, E., Strubin, M., and Hernandez, N. (1998) Genes Dev. 12, 3528-3540
44. Boyd, D. C., Greger, I. H., and Murphy, S. (2000) Gene 247, 33-44
45. Stunkel, W., Kober, I., and Seifart, K. H. (1997) Mol. Cell. Biol. 17, 4397-4405
FIGURE LEGENDS
Figure 1. Structure of the Homo sapiens (Hs), Arabidopsis thaliana (At), and Drosophila
melanogaster (Dm) snRNA promoters. See description in the text.
Figure 2. Composition of pol II and III transcription initiation complexes. A). pol II initiation
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complexes assembled on a TATA-box containing mRNA core promoter and on the human U1
snRNA core promoter. B). pol III initiation complexes assembled on the human U6 snRNA
promoter and a tRNA-type promoter. The placement of hBRF, hBRFU, and hB" is arbitrary. A
stippled line separates TBP and hBRF to indicate that these factors are tightly associated with
each other in solution. In contrast, there is no evidence that TBP and hBRFU are associated with
each other in solution.
Figure 3. Assembly of a stable snRNA transcription initiation complex. A. TBP and SNAPc
contain built-in dampers of DNA binding that downregulate their binding to DNA. B.
Assembly of the U6 initiation complex. See text for description.
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DSE PSE
TATADSE PSE
33-34
23-24
USE TATA
USE TATA
PSEA PSEB
ga G
PSEA TATA
TC
pol II
pol III
pol II
pol III
pol II
pol III
Hs
At
Dm
CORE PROMOTER REGION
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IIA
IIA
IIE
IIFIIB
Pol 2TBP
TATA
SNAP c
PSEU1
mRNA
IIH
?TBP
IIE
IIFIIB
Pol 2
IIH
TFIIIC
A B
TFIIIB
TBPhBRF
hB"
tRNA-type
SNAP c
PSE TATA
TBPhBRFU
hB"
U6
pol lll
pol ll
A
B
TAF II s
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OCTAPOUH
POUS
45
19 43
50
190
Q
PSE
OIR
TATA
TBP
190
50
19 43TATA
TBP
Q
PSE
45
OIR
A
B
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Nouria HernandezsnRNA genes: a model system to study fundamental mechanisms of transcription
published online June 4, 2001J. Biol. Chem.
10.1074/jbc.R100032200Access the most updated version of this article at doi:
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