0
1992 Elscvicr
GENE
Science
Publishcrs
B.V. All rights reacrvcd.
149
0378-l 119~92,‘SO5.00
(36508
Nucleotide (Dihydroorotic DNA)
gene of Saccharomyces
sequence of the URAl acid dehydrogenase
activity;
pyrimidine
pathway;
pyrimidine
pathway
cerevisiae*7t
regulator
1 control;
recombinant
Anne Roy C.h’.R. S.
L’. P. R. Y(103 Cu~hrog~r~Psr
Rcccived
b! J.-P. Lccocq
et Mutcrg&.rr
(W. Gqbnlski):
Mol&~laire er Stnrctu~ule. Irtsritur de Bidogie Molhlrrire
I I December
1991; Reviscd/4ccepted:
18 Februar)
et Celluhre.
6 71184 Strmhour~y Cede.\,
1992: RcceiLed at publishers:
16 March
Frcutrr~w
1992
SUMMARY
The complete nucleotide sequence of the URAl gene encoding dihydroorotic acid dehydrogcnase (DHOdehase) is presented. This enzyme catalyses the conversion of DHO to erotic acid and plays a major role in the pyrimidine pathway. as DHO is the effector of the positive control of the transcription of at least four genes, URAl, URA3, URA4 and URAfO. Comparisons between the amino acid sequence of the yeast DHOdehase and sequences of DHOdehases previously isolated from Dict~wtellum discoi’dum, Escherichia cnli and Bacillus suhtilis reveal no obvious homologies.
The URAI gene of Saccharom)ces cerevisiue encodes the dihydroorotic acid dehydrogenase (DHOdehase), which catalyses the conversion of dihydroorotic acid to erotic acid in the pyrimidine pathway (Lacroute, 1968). URAl expression is regulated at the transcriptional level through a positive control which requires both PPRl (pyrimidine pathway regulator l), the product of the regulatory PPRl gene (Loison et al., 1981; Losson and Lacroutc, 1983) and the inducer DHO (Lacroute, 1968). The induced and noninduced tsp were previously located both in the S. cereviske
Correspondertc~e to: Dr. A. Roy.
MolCculairc
ct Structurale.
hourg Cedex, France. *On rcqucst
C.P.R. Canc&gCn&e et Mutag&& I.B.M.C., 15, Rue Descartes 67084 Stras-
Tel. (33).(88).41-70-79;
the author will supply dctailcd
Fax (33)-(88).61-06-80. cxpcrimcntal
evidence
for the
conclusions reached in this Brief Note. ’ This paper IS dcdicatcd to the mernorq of Dr. J.-P. Lecocq. Ahhrcviations:
aa, amino acid(s);
acid; DHOdchasc,
bp, base pair(s):
DHO dehydrogenasc:
DHO.
kb. kilobase
dihydroorotic or 1000 bp: nt.
nucleotide( s); ORF. open reading frame; PPR I, pyrimidine pathway rcgulator I: fv/J, transcription start point(a): C,dS,,,,,. upstrcnm activating scqucncc
of PPRI-rcgnlated
gcncs;
C’RAI.
gene encoding
DHOdehnse.
background and in Schi~osac,charonz),ce.r pomhe (Losson et al., 1985). We sequenced a 2884-bp DNA fragment carrying this gene (Loison et al., 1981) by the dideoxy chain termination method (Sanger et al., 1977). Upstream (205-2 18 bp and 234-249 bp) from the tsp are protein. two binding sites (UAS,,,,, ) of a PPRl-dependent These sites, identified both by correlation to the consensus sequence and by in vitro DNase I footprinting with yeastcell extracts overproducing or not PPRl (Roy et al., 1990), are located in a 2X5-bp DNA fragment upstream from a HirrdIII site (Fig. 1) and are necessary for PPRl induction as shown by DHOdehase activity (data not shown), contrary to the previous results of Loison et al. (1981). An ORF encoding the complete 3 14-aa sequence of the dihydroorotic acid dehydrogenase is found downstream from these tsp. No special features have been noticed in the aa sequence. The hydrophobicity computed from the aa scquence according to the coefficients of Kyte and Doolittle (1982) reveals no unusual pattern (data not shown). The codon bias index calculated as suggested by Bennetzcn and Hall (1982) has a value of -0.013. Comparisons between the S. cerevisiae DHOdehase aa sequence and either the D. discoi’dum or E. coli aa sequences previously reported
150 120 240 360 CG
T'ZAMZITCCGTACCPAACATGACAGCCAGI?TPACTAC MT
AS
CCGGTGITCAT~~A~~~~A~T~~A~~~~~ GVH
CM
TTQ
E
L
D
L
TT
K
CTAT~T~T F
L
N
NTY
E
N
P
FMN
AS
TCITA~GGTAA~~ACA E
LA
NS
KAGA
F
I
T
KSA
TT
L
E
R
EG
N
PEP
RY
I
TTTCTGI%~CTAGGCAGTATCAACTCCAT~~A~A~~A~~~~~~A~~~~~A~T SV
P
LGS
I
NS
MG
L
PN
E
CAGFTWZX&TA!XAG%TIMlXWZJA~~~ G VAG M S I D EN LN L LR K TM;CTTATGACTIXACIVXCkWiCQ&FC~~~CAAAAAACCI AYD
FD
LT
K
ET
LE
KV
FA
G
I
DY
Y
LS
YV
LN
R
OK
NY
P
DA
PA
I
F
F
S
A l
QD
S
E
FN
G
I
T
E
LN
L
SC
P
czTtxrGmGTr_m F
F
KK
(Larsen and Jensen. 1985), or a B. suhtiliscluster of p~‘r genes (EMBL data library) reveal no overall significant homologies.
P
LGV
K
L
NVP
G
P
PY
F
D
FA
H
~ew~~~titrr. J. Bacterial.
FD
IMAK
106 (1968) 519-522.
Larsen, J.N. and Jensen, K.F.: taw dchydrogenasc.
Nucteotldc
transcrlplional
regulalion
struCLur;IIand
Genel. 3
acid dchqdrogcnaae
in
(19XI) I IY- 123.
F.: Plasmida carrying the gust decarho\qtase
rcgulatorq ycncs: transcription
rcgulnlion in ;L foreign
Cell 32 (1983) 371-377.
Losson. R.. Fuchs. R.P.P. and Lacroute,
C’Rj13. I!umplcs Ro>, i-\.. Ixinger,
151 (1985) 59-M
M. and Lacroutc. F.. Evidcncc TOI
of dihqdroorotic
S~cr,llrrv~,,rr?.crr crrr~i.sitrr.Curr. Loason. R. and Lacroute,
acquencc of the pl’rf) gcns 01
of the Ravoprotcin dihydrooro-
ELII-. J. Biochcm.
Loison. Cr., Jund, R., Nguqcn-Juillerct,
cnvironmcnt.
REFERENCES
V
Lacroutc, F.: Rcgula~ion of the pyrimidmc biosynthesis in ,Str~~htrro,,t~[,~,\
E‘.w/wrid~itr cc,/; and characterization
Snecial thanks arc due to F. Lacroute for his constant interest and to B. Winsor for her English editing of the manuscript. This work benefited from technical advice from F. Exinger for DHOdehase activity assay.
KPQ
GCA-
480 -22 600 -62 720 -102 840 .I42 960 -182
F.: Yeast promoters bRAI
of positixc control. J. Mol. Biol. IX5
F. and Loaaon. R.: ci\- and rrtr/wacting
( 19X5)
and
65-X1.
regulator)
cl-
cmcnts of the ycasL CR.41 promoter. Mot. Cclt. Biot. IO ( 1090) 5257Bcnnctlcn.
J.L. and Hall, B: Codon sctcclion m jeaht. J. Biol. Chcm
257
( 1’982)3026-303 I. Kytc, J. and Doohttlc,
5270. Sangcr. F.. Nlcklcn.
R.F.: L\ simple method for displaying the hydro-
pathic chxruclcr of a protein. J. Mot. Biol. 157 (1982)
105- 132.
S. and Coutson.
A.R.: DNA
sequencmg \vith chun-
tcmminating inhibitors. Proc. NatI. Acad. Sci. USA 5467.
71 (1977) 5463-
1992 Elscvicr
GENE
Science
Publishcrs
B.V. All rights reacrvcd.
149
0378-l 119~92,‘SO5.00
(36508
Nucleotide (Dihydroorotic DNA)
gene of Saccharomyces
sequence of the URAl acid dehydrogenase
activity;
pyrimidine
pathway;
pyrimidine
pathway
cerevisiae*7t
regulator
1 control;
recombinant
Anne Roy C.h’.R. S.
L’. P. R. Y(103 Cu~hrog~r~Psr
Rcccived
b! J.-P. Lccocq
et Mutcrg&.rr
(W. Gqbnlski):
Mol&~laire er Stnrctu~ule. Irtsritur de Bidogie Molhlrrire
I I December
1991; Reviscd/4ccepted:
18 Februar)
et Celluhre.
6 71184 Strmhour~y Cede.\,
1992: RcceiLed at publishers:
16 March
Frcutrr~w
1992
SUMMARY
The complete nucleotide sequence of the URAl gene encoding dihydroorotic acid dehydrogcnase (DHOdehase) is presented. This enzyme catalyses the conversion of DHO to erotic acid and plays a major role in the pyrimidine pathway. as DHO is the effector of the positive control of the transcription of at least four genes, URAl, URA3, URA4 and URAfO. Comparisons between the amino acid sequence of the yeast DHOdehase and sequences of DHOdehases previously isolated from Dict~wtellum discoi’dum, Escherichia cnli and Bacillus suhtilis reveal no obvious homologies.
The URAI gene of Saccharom)ces cerevisiue encodes the dihydroorotic acid dehydrogenase (DHOdehase), which catalyses the conversion of dihydroorotic acid to erotic acid in the pyrimidine pathway (Lacroute, 1968). URAl expression is regulated at the transcriptional level through a positive control which requires both PPRl (pyrimidine pathway regulator l), the product of the regulatory PPRl gene (Loison et al., 1981; Losson and Lacroutc, 1983) and the inducer DHO (Lacroute, 1968). The induced and noninduced tsp were previously located both in the S. cereviske
Correspondertc~e to: Dr. A. Roy.
MolCculairc
ct Structurale.
hourg Cedex, France. *On rcqucst
C.P.R. Canc&gCn&e et Mutag&& I.B.M.C., 15, Rue Descartes 67084 Stras-
Tel. (33).(88).41-70-79;
the author will supply dctailcd
Fax (33)-(88).61-06-80. cxpcrimcntal
evidence
for the
conclusions reached in this Brief Note. ’ This paper IS dcdicatcd to the mernorq of Dr. J.-P. Lecocq. Ahhrcviations:
aa, amino acid(s);
acid; DHOdchasc,
bp, base pair(s):
DHO dehydrogenasc:
DHO.
kb. kilobase
dihydroorotic or 1000 bp: nt.
nucleotide( s); ORF. open reading frame; PPR I, pyrimidine pathway rcgulator I: fv/J, transcription start point(a): C,dS,,,,,. upstrcnm activating scqucncc
of PPRI-rcgnlated
gcncs;
C’RAI.
gene encoding
DHOdehnse.
background and in Schi~osac,charonz),ce.r pomhe (Losson et al., 1985). We sequenced a 2884-bp DNA fragment carrying this gene (Loison et al., 1981) by the dideoxy chain termination method (Sanger et al., 1977). Upstream (205-2 18 bp and 234-249 bp) from the tsp are protein. two binding sites (UAS,,,,, ) of a PPRl-dependent These sites, identified both by correlation to the consensus sequence and by in vitro DNase I footprinting with yeastcell extracts overproducing or not PPRl (Roy et al., 1990), are located in a 2X5-bp DNA fragment upstream from a HirrdIII site (Fig. 1) and are necessary for PPRl induction as shown by DHOdehase activity (data not shown), contrary to the previous results of Loison et al. (1981). An ORF encoding the complete 3 14-aa sequence of the dihydroorotic acid dehydrogenase is found downstream from these tsp. No special features have been noticed in the aa sequence. The hydrophobicity computed from the aa scquence according to the coefficients of Kyte and Doolittle (1982) reveals no unusual pattern (data not shown). The codon bias index calculated as suggested by Bennetzcn and Hall (1982) has a value of -0.013. Comparisons between the S. cerevisiae DHOdehase aa sequence and either the D. discoi’dum or E. coli aa sequences previously reported
150 120 240 360 CG
T'ZAMZITCCGTACCPAACATGACAGCCAGI?TPACTAC MT
AS
CCGGTGITCAT~~A~~~~A~T~~A~~~~~ GVH
CM
TTQ
E
L
D
L
TT
K
CTAT~T~T F
L
N
NTY
E
N
P
FMN
AS
TCITA~GGTAA~~ACA E
LA
NS
KAGA
F
I
T
KSA
TT
L
E
R
EG
N
PEP
RY
I
TTTCTGI%~CTAGGCAGTATCAACTCCAT~~A~A~~A~~~~~~A~~~~~A~T SV
P
LGS
I
NS
MG
L
PN
E
CAGFTWZX&TA!XAG%TIMlXWZJA~~~ G VAG M S I D EN LN L LR K TM;CTTATGACTIXACIVXCkWiCQ&FC~~~CAAAAAACCI AYD
FD
LT
K
ET
LE
KV
FA
G
I
DY
Y
LS
YV
LN
R
OK
NY
P
DA
PA
I
F
F
S
A l
QD
S
E
FN
G
I
T
E
LN
L
SC
P
czTtxrGmGTr_m F
F
KK
(Larsen and Jensen. 1985), or a B. suhtiliscluster of p~‘r genes (EMBL data library) reveal no overall significant homologies.
P
LGV
K
L
NVP
G
P
PY
F
D
FA
H
~ew~~~titrr. J. Bacterial.
FD
IMAK
106 (1968) 519-522.
Larsen, J.N. and Jensen, K.F.: taw dchydrogenasc.
Nucteotldc
transcrlplional
regulalion
struCLur;IIand
Genel. 3
acid dchqdrogcnaae
in
(19XI) I IY- 123.
F.: Plasmida carrying the gust decarho\qtase
rcgulatorq ycncs: transcription
rcgulnlion in ;L foreign
Cell 32 (1983) 371-377.
Losson. R.. Fuchs. R.P.P. and Lacroute,
C’Rj13. I!umplcs Ro>, i-\.. Ixinger,
151 (1985) 59-M
M. and Lacroutc. F.. Evidcncc TOI
of dihqdroorotic
S~cr,llrrv~,,rr?.crr crrr~i.sitrr.Curr. Loason. R. and Lacroute,
acquencc of the pl’rf) gcns 01
of the Ravoprotcin dihydrooro-
ELII-. J. Biochcm.
Loison. Cr., Jund, R., Nguqcn-Juillerct,
cnvironmcnt.
REFERENCES
V
Lacroutc, F.: Rcgula~ion of the pyrimidmc biosynthesis in ,Str~~htrro,,t~[,~,\
E‘.w/wrid~itr cc,/; and characterization
Snecial thanks arc due to F. Lacroute for his constant interest and to B. Winsor for her English editing of the manuscript. This work benefited from technical advice from F. Exinger for DHOdehase activity assay.
KPQ
GCA-
480 -22 600 -62 720 -102 840 .I42 960 -182
F.: Yeast promoters bRAI
of positixc control. J. Mol. Biol. IX5
F. and Loaaon. R.: ci\- and rrtr/wacting
( 19X5)
and
65-X1.
regulator)
cl-
cmcnts of the ycasL CR.41 promoter. Mot. Cclt. Biot. IO ( 1090) 5257Bcnnctlcn.
J.L. and Hall, B: Codon sctcclion m jeaht. J. Biol. Chcm
257
( 1’982)3026-303 I. Kytc, J. and Doohttlc,
5270. Sangcr. F.. Nlcklcn.
R.F.: L\ simple method for displaying the hydro-
pathic chxruclcr of a protein. J. Mot. Biol. 157 (1982)
105- 132.
S. and Coutson.
A.R.: DNA
sequencmg \vith chun-
tcmminating inhibitors. Proc. NatI. Acad. Sci. USA 5467.
71 (1977) 5463-
