6736 Nucleic Acids Research, 1992, Vol. 20, No. 24
.:II/ 1992 Oxford University Press
Elucidation of three putative structural subdomains by comparison of primary structure of Xenopus and human RAP74 Da-Wei Gong, Satoshi Hasegawa2, Keiji Wada1, Robert G.Roeder2, Yoshihiro Nakatani and Masami Horikoshi2'3' * Laboratory of Molecular Biology, NINDS and 'Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, MD 20892, 2Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021 and 3Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan EMBL accession no. Z17426
Received November 9, 1992; Accepted November 13, 1992 RAP74 is a component of TFIIF, a transcription factor that interacts with RNA polymerase II (1) and is essential for its recruitment to the preformed TFIID-TFILB-promoter complex (2). TFIIF is a tetramer of two RAP30 and two RAP74 subunits. The protein is multifunctional, playing an important role in transcription elongation as well as in initiation (3). A structure-function analysis of TFIIF should thus provide insight into the relationship between transcription initiation and elongation. Recently, the sequence of a human cDNA encoding RAP74 was obtained and revealed the presence of several interesting structural features of the protein (4, 5). The central region contains a high content of charged residues, while the rest appears to have a globular structure. Furthermore, the C-terminal segment contains sigma-similarity region (S.Hashimoto, R.G.R. and M.H., unpublished results). Here we report the cDNA cloning of Xenopus RAP74 and compare its primary structure to that of the human RAP74 (Figure 1). Xenopus RAP74 cDNAs were isolated by screening a Xenopus oocyte cDNA library under low stringency conditions using human RAP74 cDNA as a probe. The deduced amino acid Xenopus Human
1 1
51 51 101 101 151 151 201 197 251 243 301 296 351 346 401 396 451 444
NaSLOTSOOS VTHTVVRVPR NPHXRYSLMA FHAADXVDFS TWNQRHIURD a
K
P 8 N
*
T
I
L
A
RxKY cXLRT v X
T
Hav=¢vTASYFIFTQC ADGoFKArPV
RVNG
T
P
Y
L
RPZ
L
L
KGOKGKIOCK XKSDLKIRDLZ DDLZLSSTZS KNSDZZGOSR KKPQKKVH AH R N DA Da G GRV P As AY C0IG1KKI¶~D DDALHDSDW DrOQE VDDYN SDZSSSDZL rCXIE a
r
SQ Z
100
NFYPTA 150
H H
R -----RR
RXd
50 50
FVDDOWIL 100
MlTLTAHAk HQnHRRHV LNHHTINQQR RLKDQVGDED ZDHHGGOKLH H S ---- Q DEH K R
aaQ
150 200 196 250 242 300 295
ZGPKGLDHQS HSSHZZHK AZX ZHHK KAPTPQDHXK XKKKHDS8 350 V
HKt X R
PP
D
R --
H
345
HTs DSDIDOASSLFNOQKK KTPPKKDXUG GONHHRHGS RPGTPHPDTG 400 DH H s Ar A 395 R R - P OG AZG NTSSTLRAaA SKLHQHGT VSNTPAAXRL 0
s
SN
--
AGEPQNTS GSTPQQPQSG
RLDT
HL
P
KSTPSHGDIQ LTZEAVRRYL TRXPTTIKDL LxxrQTKKTT LS88QTVmvL T
N
V V D
501 AQILKHIPD RKVVHDIOV YLKH
494
HA
ST XNI
WSG&GSH YHRXQRHHR
SaQ
z
MIH
8
450 443 500
493
524
517
Figure 1. Amino acid sequence of Xenopus laevis RAP74 and its comparison with human RAP74. Amino acid sequences of Xenopus and human RAP74 are shown in the first and second lines, respectively. For human RAP74, only the amino acids different from Xenopus RAP74 are shown. The sigma-similarity region is indicated as a box. *
To whom correspondence should be addressed
sequence of Xenopus RAP74 shows 76% identity and 86% similarity to the human sequence. The overall structure apparently consists of three domains based on both conservation and continuity of amino acid sequence between Xenopus and human RAP74. Those regions also differ markedly in the number of amino acid insertion and deletion sites present. The N-terminal segment (amino acids 1-185) has none, the highly charged central domain (amino acids 186-380) contains 9 insertion/deletion sites, and the C-terminal region (amino acids 381-517) contains only one. Interestingly, 8 out of 10 of the insertion/deletion sites contain glycine residues, and the other two have glycine within the next two flanking amino acids. The presence of these glycine residues may serve as a flexible joint in this multifunctional TFIIF protein. The data on the sequence of Xenopus RAP74 should be useful for additional structural and functional studies.
ACKNOWLEGEMENTS We thank Dr Mike Brenner for a critical reading of the manuscript and Drs Jerry Thomsen, Doug A.Melton and Bruce Blumberg for the Xenopus laevis cDNA library and Drs Sherman M.Weissman, Shigetaka Kitajima and Yukio Yasukochi for the human RAP74 cDNA clone. M.H. was an Alexandrine and Alexander L.Sinsheimer Scholar. A part of this study was supported by grants from the National Institutes of Health to R.G.R. and M.H. and general support by the Pew Trusts to The Rockefeller University.
REFERENCES 1. Sopta,M., Carthew,R.W. and Greenblatt,J. (1985) J. Biol. Chem. 260, 10353-10361. 2. Maldonado,E., Ha,I., Cortes,P., Weis,L. and Reinberg,D. (1990) Mol. Cell. Biol. 10, 6335-6347. 3. Flores,O., Maldonado,E. and Reinberg,D. (1989) J. Biol. Chem. 264, 8913 -8921. 4. Aso,T., Vasavada,A., Kawaguchi,T., Geronimo,F.J., Ganguly,S., Kitajima,S., Weissman,S.M. and Yasukochi,Y. (1992) Nature 355, 461-464. 5. Finkelstein,A., Kstrub,C.P., Li.J., Chavez,D.P., Wang,B.Q., Fang,S.M., Greenblatt,J. and Burton,Z.F. (1992) Nature 355, 464-467.
.:II/ 1992 Oxford University Press
Elucidation of three putative structural subdomains by comparison of primary structure of Xenopus and human RAP74 Da-Wei Gong, Satoshi Hasegawa2, Keiji Wada1, Robert G.Roeder2, Yoshihiro Nakatani and Masami Horikoshi2'3' * Laboratory of Molecular Biology, NINDS and 'Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, MD 20892, 2Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021 and 3Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan EMBL accession no. Z17426
Received November 9, 1992; Accepted November 13, 1992 RAP74 is a component of TFIIF, a transcription factor that interacts with RNA polymerase II (1) and is essential for its recruitment to the preformed TFIID-TFILB-promoter complex (2). TFIIF is a tetramer of two RAP30 and two RAP74 subunits. The protein is multifunctional, playing an important role in transcription elongation as well as in initiation (3). A structure-function analysis of TFIIF should thus provide insight into the relationship between transcription initiation and elongation. Recently, the sequence of a human cDNA encoding RAP74 was obtained and revealed the presence of several interesting structural features of the protein (4, 5). The central region contains a high content of charged residues, while the rest appears to have a globular structure. Furthermore, the C-terminal segment contains sigma-similarity region (S.Hashimoto, R.G.R. and M.H., unpublished results). Here we report the cDNA cloning of Xenopus RAP74 and compare its primary structure to that of the human RAP74 (Figure 1). Xenopus RAP74 cDNAs were isolated by screening a Xenopus oocyte cDNA library under low stringency conditions using human RAP74 cDNA as a probe. The deduced amino acid Xenopus Human
1 1
51 51 101 101 151 151 201 197 251 243 301 296 351 346 401 396 451 444
NaSLOTSOOS VTHTVVRVPR NPHXRYSLMA FHAADXVDFS TWNQRHIURD a
K
P 8 N
*
T
I
L
A
RxKY cXLRT v X
T
Hav=¢vTASYFIFTQC ADGoFKArPV
RVNG
T
P
Y
L
RPZ
L
L
KGOKGKIOCK XKSDLKIRDLZ DDLZLSSTZS KNSDZZGOSR KKPQKKVH AH R N DA Da G GRV P As AY C0IG1KKI¶~D DDALHDSDW DrOQE VDDYN SDZSSSDZL rCXIE a
r
SQ Z
100
NFYPTA 150
H H
R -----RR
RXd
50 50
FVDDOWIL 100
MlTLTAHAk HQnHRRHV LNHHTINQQR RLKDQVGDED ZDHHGGOKLH H S ---- Q DEH K R
aaQ
150 200 196 250 242 300 295
ZGPKGLDHQS HSSHZZHK AZX ZHHK KAPTPQDHXK XKKKHDS8 350 V
HKt X R
PP
D
R --
H
345
HTs DSDIDOASSLFNOQKK KTPPKKDXUG GONHHRHGS RPGTPHPDTG 400 DH H s Ar A 395 R R - P OG AZG NTSSTLRAaA SKLHQHGT VSNTPAAXRL 0
s
SN
--
AGEPQNTS GSTPQQPQSG
RLDT
HL
P
KSTPSHGDIQ LTZEAVRRYL TRXPTTIKDL LxxrQTKKTT LS88QTVmvL T
N
V V D
501 AQILKHIPD RKVVHDIOV YLKH
494
HA
ST XNI
WSG&GSH YHRXQRHHR
SaQ
z
MIH
8
450 443 500
493
524
517
Figure 1. Amino acid sequence of Xenopus laevis RAP74 and its comparison with human RAP74. Amino acid sequences of Xenopus and human RAP74 are shown in the first and second lines, respectively. For human RAP74, only the amino acids different from Xenopus RAP74 are shown. The sigma-similarity region is indicated as a box. *
To whom correspondence should be addressed
sequence of Xenopus RAP74 shows 76% identity and 86% similarity to the human sequence. The overall structure apparently consists of three domains based on both conservation and continuity of amino acid sequence between Xenopus and human RAP74. Those regions also differ markedly in the number of amino acid insertion and deletion sites present. The N-terminal segment (amino acids 1-185) has none, the highly charged central domain (amino acids 186-380) contains 9 insertion/deletion sites, and the C-terminal region (amino acids 381-517) contains only one. Interestingly, 8 out of 10 of the insertion/deletion sites contain glycine residues, and the other two have glycine within the next two flanking amino acids. The presence of these glycine residues may serve as a flexible joint in this multifunctional TFIIF protein. The data on the sequence of Xenopus RAP74 should be useful for additional structural and functional studies.
ACKNOWLEGEMENTS We thank Dr Mike Brenner for a critical reading of the manuscript and Drs Jerry Thomsen, Doug A.Melton and Bruce Blumberg for the Xenopus laevis cDNA library and Drs Sherman M.Weissman, Shigetaka Kitajima and Yukio Yasukochi for the human RAP74 cDNA clone. M.H. was an Alexandrine and Alexander L.Sinsheimer Scholar. A part of this study was supported by grants from the National Institutes of Health to R.G.R. and M.H. and general support by the Pew Trusts to The Rockefeller University.
REFERENCES 1. Sopta,M., Carthew,R.W. and Greenblatt,J. (1985) J. Biol. Chem. 260, 10353-10361. 2. Maldonado,E., Ha,I., Cortes,P., Weis,L. and Reinberg,D. (1990) Mol. Cell. Biol. 10, 6335-6347. 3. Flores,O., Maldonado,E. and Reinberg,D. (1989) J. Biol. Chem. 264, 8913 -8921. 4. Aso,T., Vasavada,A., Kawaguchi,T., Geronimo,F.J., Ganguly,S., Kitajima,S., Weissman,S.M. and Yasukochi,Y. (1992) Nature 355, 461-464. 5. Finkelstein,A., Kstrub,C.P., Li.J., Chavez,D.P., Wang,B.Q., Fang,S.M., Greenblatt,J. and Burton,Z.F. (1992) Nature 355, 464-467.
