|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 40, 31266-31268, October 6, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, March 27, 2000, and in revised form, April 28, 2000
TFIIS is a transcription elongation factor that
consists of three domains. We have previously solved the structures of
domains II and III, which stimulate arrested polymerase II elongation complexes in order to resume transcription. Domain I is conserved in
evolution from yeast to human species and is homologous to the
transcription factors elongin A and CRSP70. Domain I also interacts with the transcriptionally active RNA polymerase II holoenzyme and therefore, may have a function unrelated to the previously described transcription elongation activity of TFIIS. We
have solved the structure of domain I of yeast TFIIS using NMR
spectroscopy. Domain I is a compact four-helix bundle that is
structurally independent of domains II and III of the TFIIS. Using the
yeast structure as a template, we have modeled the homologous domains
from elongin A and CRSP70 and identified a conserved positively charged
patch on the surface of all three proteins, which may be involved in
conserved functional interactions with the transcriptional machinery.
TFIIS1 is a
transcription elongation factor that increases the overall
transcription rate of RNA polymerase II by reactivating transcription
elongation complexes that have arrested transcription (1). TFIIS is
conserved from yeast to man, and homologs are found in Archaea and in
some viral genomes. TFIIS comprises three structural domains termed I,
II, and III. A fragment consisting of domains II and III is sufficient
for elongation activity in vitro and is able to rescue the
phenotype of a TFIIS gene disruption in yeast cells. Domain II
(residues 131-240) mediates the interaction with the largest subunit
of RNA polymerase. Domain III (residues 264-309), a small
zinc-ribbon motif, is implicated in the stimulation of
transcript cleavage and resumption of transcription by RNA polymerase
II (2, 3).
Several observations implicate the N-terminal domain of TFIIS (domain
I) in the transcription process. First, domain I interacts directly
with the RNA polymerase II holoenzyme and can be used to purify the
holoenzyme using protein affinity chromatography (4). Second, domain I
is conserved from yeast to human and is also homologous to regions of
elongin A (5) and CRSP70 (6), both of which are involved in
transcription. Elongin is a transcription elongation factor that
increases the rate of transcription by suppressing transient pausing of
the elongation complex (7). CRSP70 is an essential subunit of the CRSP
complex, which is required for the activity of the enhancer-binding
protein Sp1 (4). Third, in Saccharomyces cerevisiae, TFIIS
lacking domain I is synthetically lethal with a disruption of the
TFG3 gene. Domains II and III, which are sufficient to
reactivate arrested transcription complexes, are not able to complement
the synthetic lethality with
TFG3.2 TFG3 is a component of
the general transcription factors TFIIF and TFIID and swi/SNF, a
complex required for full activity of several transcription activators
(8).
Thus, biochemical and genetic studies of the N terminus of TFIIS
suggest a role in transcription. As yet, the functions of the
N-terminal domain of TFIIS, elongin, or CRSP70 are not known. To help
elucidate the function of this domain and to provide a structural
framework for the design and interpretation of additional studies, we
determined the solution structure of domain I of S. cerevisiae TFIIS and used this structure to model the N-terminal domains of elongin A, and CRSP70.
Cloning, Expression, and Purification of TFIIS--
Sequences
coding for residues 1 to 111 of yeast (S. cerevisiae) TFIIS
were cloned into the T7 polymerase expression vector pET15b (Novagen),
as described in Morin et al. (9) and expressed as C-terminal
fusions to an N-terminal His6 tag and a thrombin protease
site. Escherichia coli BL21(DE3) cells expressing the TFIIS
fragment were grown in M9 minimal medium (3) containing 15N-labeled NH4Cl and for
13C-labeled samples, 13C-labeled glucose. Cells
were grown at 30 °C to an absorbance of 0.4 at 600 nm, and
protein expression was induced with 1.0 mM
isopropyl-6-D-thiogalactopyranoside. Three hours after
induction, the cells were harvested by centrifugation and resuspended
in Buffer A (30 mM Hepes, 600 mM NaCl, 10 µM ZnCl, 1.0 mM benzamidine, pH 7.5) with 5 mM imidazole and frozen at NMR Spectroscopy and Spectral Assignments--
All NMR spectra
were collected at 25 °C on either a Varian 500-MHz or 600-MHz Inova
spectrometer equipped with pulse field gradient units and actively
shielded z-gradient triple-resonance probes. NMR experiments involving
correlations of amide protons were acquired with gradient enhanced
versions (10) of the originally published pulse sequences. NMR data
were processed using nmrPipe software (11). Spectral analysis was
assisted using the programs Pipp and Capp (12) and NMRView (13).
Sequence-specific assignment of 1HN, 15N,
13C Structure Determination--
Structure calculations were
performed using version 3.851 of XPLOR with ambiguous restraints for
iterative assignment (ARIA) (24). The initial input for ARIA consisted
of the eight lowest energy structures calculated by XPLOR using 276 unambiguous manually assigned NOEs, dihedral angle restraints, and
hydrogen bond restraints. The dihedral restraints were based on the
predictions of TALOS (25). All TALOS-derived restraints were consistent
with the secondary structure as determined from
3JNH-H
The CN-NOESY, 15N-edited NOESY-HSQC, and homonuclear
two-dimensional NOESY were automatically peak picked using NMRView (13) followed by manual removal of obvious artifacts. The resulting NOE peak
lists were used as input for ARIA calculations. The frequency window
tolerance for assigning NOEs with ARIA was ±0.03 ppm for all proton
dimensions and ±0.5 ppm in the nitrogen and carbon dimensions. The
ARIA parameters p, Tv and
Nv were as in Nilges et al. (24). In
each iteration, 20 structures were refined, and the 7 lowest energy
structures were used for the purposes of NOE assignments. In the final
(eighth) ARIA iteration, 20 structures were refined, and the 10 lowest
energy structures were retained for analysis. A total of 1199 unambiguous and 542 ambiguous NOE-based distance restraints were used
for the final set of structures.
Homology modeling of human TFIIS, elongin A, and CRSP70 using the yeast
structure as a template was performed using the "optimize" option
of SWISS-MODEL Version 3.5 (26). The sequence alignment was generated
using the multiple sequence alignment program Clustal W1.8 (27) and
edited slightly by hand to optimize the alignment over the helices in
favor of aligning over the loops. Surface charge plots were produced
using GRASP (28).
Partial proteolysis studies revealed that the N-terminal region of
yeast TFIIS consists of a stable structural domain. This domain
extended from the N terminus of TFIIS to the region between residues
105 and 124 (9). To identify the fragment of domain I most suitable for
structural studies, three TFIIS constructs containing residues 1-93,
1-111, and 1-124 were prepared, and the resulting
15N-labeled proteins tested for feasibility by NMR. The
TFIIS-(1-111) construct containing the conserved residues was
expressed well, was stable for several weeks, and had all the dispersed
HSQC peaks (characteristic of residues in structured parts of the
protein) that were observed in the longer TFIIS-(1-124) construct. The TFIIS-(1-111) construct was selected for structure determination.
Domain I of TFIIS formed a four-helix bundle (Fig.
1). The helices are each 12 to 13 residues long and are connected by structured loops of length 4, 9, and
6 residues. Residues 78-111 are unstructured. The structure of domain
I is likely to be conserved in elongin and CRSP70. The hydrophobic core
residues of helices 2, 3, and 4 are well conserved among TFIIS,
elongin, and CRSP70, although helix 1 is less conserved (Fig.
2). The DALI data base (29) revealed that
the closest structural homolog is the A chain of cytochrome
c oxidase (Z-score = 4.1, root mean squared
deviation (RMSD) = 2.7), which is also a four-helix bundle
although its helices are much longer than those of TFIIS.
The surfaces of the proteins were examined to identify regions that
might participate in functional interactions. The structures of
proteins were modeled with SWISS-MODEL Version 3.5 (26) using the yeast
TFIIS structure as a template. The reliability of the models depends on
the degree of sequence identity between the template and model
sequences. The models are expected to be accurate for human TFIIS and
helices 2, 3, and 4 of elongin A and CRSP70 because these helices share
high sequence homology with the TFIIS structure. The modeled structures
are likely to be poorer in helix 1 because the sequence homology in
this region is lower.
TFIIS and human elongin A both bind the transcription holoenzyme. To
indicate what part of the proteins might be responsible for binding, we
looked for surface features conserved between these two proteins (Fig.
3). Several surface residues are
conserved in TFIIS and elongin: Leu-7, Asn-12, Glu-14, Asn-19, Leu-24,
Thr-37, Leu-41, Val-46, Lys-54, Lys-55, Lys-66, Met-68, Ile-76, and
Lys-73. All of these residues with the exception of Lys-73 are also
conserved in CRSP70. Several of these residues localize to the top of
the helix bundle and the face of the protein formed by helices 1 and 3. These form a basic patch, which is most extensive in human TFIIS. The
charged nature of this conserved patch makes it a likely candidate for
the location of an interaction common to TFIIS, elongin, and CRSP70.
Other than this basic patch, the surface charge properties vary
considerably between the proteins. This may reflect binding of
different targets or may indicate that the remainder of the surface is
not functionally important. Helix 2 and 4 of CRSP70 and elongin A have
basic patches that are not found in TFIIS.
Structure of a Conserved Domain Common to the Transcription
Factors TFIIS, Elongin A, and CRSP70*
§,¶
, and**
Ontario Cancer Institute and Department of
Medical Biophysics, University of Toronto, Toronto, Ontario M5G
2M9, Canada and the ¶ C. H. Best Institute, Banting and Best
Department of Medical Research, University of Toronto, Toronto,
Ontario M5G 1L6, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
70 °C. The cells were lysed
using a French pressure cell, and the supernatant was clarified by
centrifugation at 100,000 × g for 40 min at 4 °C.
All subsequent steps were performed at 4 °C. The supernatant
solution was loaded onto a (5 × 5)-cm DE52 (Whatman, Maidstone,
UK) column, and equilibrated in Buffer A. The flow-through was loaded
onto a (1 × 4)-cm Ni2+ column and washed extensively
with Buffer A with 30 mM imidazole, and then the TFIIS was
eluted with Buffer A and 500 mM imidazole. The histidine
tag was removed by incubation with thrombin (1:1000, w/w) in 2.5 mM CaCl2, and the sample was dialyzed into NMR
buffer (10 mM NaH2PO4, 300 mM NaCl, 5 mM dithiothreitol, 50 µM EDTA, 10 µM ZnSO4, pH 7.5).
TFIIS was then further purified using gel filtration (Superdex-75
preparation grade column) and concentrated to 2 mM by
ultrafiltration. For NMR experiments requiring a D2O sample, the protein was lyophilized and redissolved in 99.9%
D2O.
and 13C
resonances for all non-proline residues of TFIIS was achieved through
correlations from CBCA(CO)NH (14), CBCANH (15), and 15N
NOESY-HSQC (16) spectra. Side chain 1H and 13C
resonances of aliphatic residues were assigned from an HCCH-TOCSY (17,
18), a CCC-TOCSY (19) and an 15N TOCSY-HSQC (20) (mixing
time, 81 ms). Homonuclear two-dimensional NOESY (21) and TOCSY (22)
spectra in D20 were used to assign side chain
1H resonances of aromatic residues. H-H nuclear Overhauser
effects (NOE) were identified from the following spectra:
13C, 15N-edited NOESY in H20 (16)
(both mixing times are 150 ms) and homonuclear NOESY in
D2O. An HNHA was used to measure
3JNH-H coupling (23).
measurements, NOE patterns, and
H and C chemical shifts. These indicated
an 
-helical conformation for residues 3-16, 20-31, 41-53, and
60-75. Hydrogen bond restraints (H···O, 2.5 Å; N···O, 3.5 Å) were added for residues which were clearly -helical as judged by
NOE patterns, chemical shift values, and lower rates of amide hydrogen exchange.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
NMR solution structure of yeast TFIIS.
The left panel shows the superposition of the backbone of
the 10 lowest energy structures. The right panel is a
Molscript (31) ribbon diagram showing the residues that form the
hydrophobic core.
View larger version (48K):
[in a new window]
Fig. 2.
Sequence alignment of TFIIS domain I. Dark gray indicates identical residues and light
gray, homologous residues. The positions of the helices are shown
above the sequence and asterisks indicate the residues that
form the hydrophobic core.
View larger version (67K):
[in a new window]
Fig. 3.
Top, GRASP (28) surface plot of yeast
TFIIS showing the conserved surface residues. Bottom, GRASP
electrostatic charge plots showing proteins oriented as in the
top center.
In vertebrates, TFIIS is expressed in several distinct isoforms where one seems to be a general form and one is testes-specific (30). One main difference between the isoforms is the length of the region linking domains I and II. Perhaps changing the length of this linker may modify the function of TFIIS by changing the configuration and orientation of proteins within TFIIS-containing complexes.
In conclusion, we have solved the structure of yeast TFIIS and shown that the overall fold of this domain is conserved among TFIIS, elongin A, and CRSP70. We modeled the homologous proteins using the yeast structure as a template and identified a conserved patch of charge, which may be involved in functional interactions common to all three proteins.
|
| |
FOOTNOTES |
|---|
* This research was funded in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1EO0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). .
The chemical shift assignments have been deposited in the BioMagResBank Database under the BMRB accession number 2583.
§ Recipient of a Studentship from the Medical Research Council of Canada.
Medical Research Council of Canada Scientist.
** To whom correspondence should be addressed: Ontario Cancer Inst. and Dept. of Medical Biophysics, University of Toronto, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Tel.: 416- 946-2017; Fax: 416- 946-6529; E-mail: carrow@oci.utoronto.ca.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M002595200
2 J. Davie and C. M. Kane, personal communication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: TFIIS, transcription factor IIS; NOESY, nuclear Overhauser spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence spectroscopy; CBCA(CO)NH, CBCANH, HCCH, CCC, and CN NMR, three-dimensional heteronuclear double and triple-resonance NMR; ARIA, ambiguous restraints for iterative assignment.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
| 1. | Reines, D. (1994) in Transcription: Mechanism and Regulation (Conaway, R. C. , and Conaway, J. W., eds), Vol. 3 , pp. 263-278, Raven Press, New York |
| 2. | Awrey, D. E., Shimasaki, N., Koth, C. K., Weilbaecher, R., Olmsted, V., Shan, X., Kazanis, S., Arellano, J., Arrowsmith, C. H., Kane, C. M., and Edwards, A. M. (1998) J. Biol. Chem. 273, 22595-22605 |
| 3. | Olmsted, V. K., Awrey, D. E., Koth, C., Shan, X., Morin, P. E., Kazanis, S., Edwards, A. M., and Arrowsmith, C. H. (1998) J. Biol. Chem. 273, 22589-22594 |
| 4. | Pan, G., Aso, T., and Greenblatt, J. (1997) J. Biol. Chem. 272, 24563-24571 |
| 5. | Shilatifard, A., Lane, W. S., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1996) Science 271, 1873-1876 |
| 6. | Ryu, S., Zhou, S., Ladurner, A. G., and Tjian, R. (1999) Nature 397, 446-450 |
| 7. | Shilatifard, A. (1998) FASEB 12, 1437-1446 |
| 8. | Kadonaga, J. (1998) Cell 92, 307-313 |
| 9. | Morin, P., Awrey, D., Edwards, A. M., and Arrowsmith, C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10604-10608 |
| 10. | Kay, L. E., Keifer, P., and Sarrinen, T. (1992) J. Am. Chem. Soc. 114, 10663-10665 |
| 11. | Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293 |
| 12. | Garrett, D. S., Powers, R., Gronenborn, A. M., and Clore, G. M. (1991) J. Magn. Reson. 95, 214-220 |
| 13. | Johnson, B. A., and Blevins, R. A. (1994) J. Biomol. NMR 4, 603-614 |
| 14. | Gronenborn, A. M., and Clore, G. M. (1996) Protein Sci. 5, 174-177 |
| 15. | Kay, L. E., Xu, G. Y., and Yamazaki, T. (1994) J. Magn. Reson. Ser. A 109, 129 |
| 16. | Pascal, S., Muhandiram, R. D., Yamazaki, T., J. D., F.-K., and Kay, L. E. (1994) J. Magn. Reson. 101, 197-201 |
| 17. | Bax, A., Clore, G. M., and Gronenborn, A. M. (1990) J. Magn. Reson. 88, 425-431 |
| 18. | Kay, L. E., Xu, G. Y., Singer, A. U., Muhandiram, D. R., and Forman-Kay, J. (1993) J. Magn. Reson. Ser. B 101, 133-136 |
| 19. | Montelione, G. T., Lyons, B. A., Emerson, S. D., and Tashiro, M. (1992) J. Am. Chem. Soc. 114, 10974 |
| 20. | Marion, D., Ikura, M., Tschudin, R., and Bax, A. (1989) J. Magn. Reson. 85, 393-399 |
| 21. | Jeener, J., Meier, B. H., Backmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546-4553 |
| 22. | Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528 |
| 23. | Kay, L. E., and Bax, A. (1990) J. Magn. Reson. 86, 110 |
| 24. | Nilges, M., Macias, M., O'Donoghue, S., and Oschkinat, H. (1997) J. Mol. Biol. 269, 408-422 |
| 25. | Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biolmol. NMR 13, 289-302 |
| 26. | Peitsh, M. C. (1996) Biochem. Soc. Trans. 24, 274-279 |
| 27. | Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G., and Gibson, T. J. (1998) Trends Biochem. Sci. 23, 403-405 |
| 28. | Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296 |
| 29. | Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138 |
| 30. | Labhart, P., and Morgan, G. T. (1998) Genomics 52, 278-288 |
| 31. | Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950 |
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H.-M. Bourbon Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex Nucleic Acids Res., July 1, 2008; 36(12): 3993 - 4008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Kim, A. I. Nesvizhskii, P. G. Rani, S. Hahn, R. Aebersold, and J. A. Ranish The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes PNAS, October 9, 2007; 104(41): 16068 - 16073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. N. Fish, M. L. Ammerman, J. K. Davie, B. F. Lu, C. Pham, L. Howe, A. S. Ponticelli, and C. M. Kane Genetic Interactions Between TFIIF and TFIIS Genetics, August 1, 2006; 173(4): 1871 - 1884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ling, A. J. Smith, and G. T. Morgan A sequence motif conserved in diverse nuclear proteins identifies a protein interaction domain utilised for nuclear targeting by human TFIIS. Nucleic Acids Res., January 1, 2006; 34(8): 2219 - 2229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Cherepanov, E. Devroe, P. A. Silver, and A. Engelman Identification of an Evolutionarily Conserved Domain in Human Lens Epithelium-derived Growth Factor/Transcriptional Co-activator p75 (LEDGF/p75) That Binds HIV-1 Integrase J. Biol. Chem., November 19, 2004; 279(47): 48883 - 48892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Smith, Y. Ling, and G. T. Morgan Subnuclear Localization and Cajal Body Targeting of Transcription Elongation Factor TFIIS in Amphibian Oocytes Mol. Biol. Cell, March 1, 2003; 14(3): 1255 - 1267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sijbrandi, U. Fiedler, and H. Th. M. Timmers RNA polymerase II complexes in the very early phase of transcription are not susceptible to TFIIS-induced exonucleolytic cleavage Nucleic Acids Res., June 1, 2002; 30(11): 2290 - 2298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Taatjes, A. M. Naar, F. Andel III, E. Nogales, and R. Tjian Structure, Function, and Activator-Induced Conformations of the CRSP Coactivator Science, February 8, 2002; 295(5557): 1058 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. B. Shimasaki and C. M. Kane Structural Basis for the Species-specific Activity of TFIIS J. Biol. Chem., November 17, 2000; 275(47): 36541 - 36549. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |