Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli

Vol. 105, No. 4, 1982 April 29, 1982

NUCLEOTIDE SEQUENCE PROTON-TRANSLOCATING

Hiroshi

Kanazawa,

16.

Tatsuya

Kayano,

Faculty

of Microbiology, Okayama University,

March

RESEARCH COMMUNICATIONS Pages 1257-1264

OF THE GENES FOR B AND E SUBUNITS ATPASE FROM ESCHERICHIA COLI

Toshiaki

Department

Received

AND BIOPHYSICAL

BIOCHEMICAL

Kiyasu

of Okayama

and

OF

Masamitsu

Pharmaceutical 700, Japan

Futai

Sciences

1982

Summary The 1855-nucleotide long DNA sequence of part of the gene cluster for the proton-translocating ATPase from E. was determined by -__ coli the method of Maxam-Gilbert. The sequence covers the genes for the fi and E subunits of Fl along with the flanking region. The amino acid sequence of these subunits deduced from the nucleotide sequence indicates that the B and E subunits have 459 and 138 amino acids, respectively. The possible secondary structure of the both subunits was estimated from the deduced primary structures. nucleotide site in A possible binding the B subunit is also discussed on the basis of the primary and secondary structures. The codons used in the genes for all the components of FlF were different in different genes, suggesting that the amount of eat R subunit in the FIFO is determined to some extent on a translational level.

Proton-translocating hydrolysis

of

portions:

ATP,

Fl,

portion

of

ATPase

the

different

subunits,

To

determine

mechanism of

attempted

to

genes

linkage of and

the c

F1

has

E.

F.

a,

and

c.

b, fine

ATP

these

map

(2-4).

genes

for

of we except

DNA

F

0 could the

6

and

of

its

83

minute

part

of

the

part the

E subunits

primary and

Fl and Fo, Abbreviations used: peripheral proton-translocating ATPase, respectively; carbodiimide; a, b, and c subunits, the decreasing molecular weight.

the

of

of

different has

E.

determine

the

structure

of

the

three

coli

and

primary FIFO,

we

We showed

that

all

of

E.

coli

region

the

nucleotide of

B and structure

integral DCCD, subunits of

different

five

FIFO

and

membrane

and

determine

and

two

integral has

B subunit of

of

an

genes.

all

synthesis

channel

primary

determined

major

0' and

to

the

the

determine 4 and

proton

components

sequence

we

F

essential

deduce

the

composed

site a

is

at

except

is and

catalytic

of

located

and

enzyme

forms

it

Previously, ~1, y

subunits

a

To

the are

catalyzes

portion,

structur,e

components.

FIFO

The

synthesis,

determine

for

Consequently, components

(1).

6 and

the of

structure

the

y,

coli

E. -__

membrane

complex.

~1, B,

the

reversibly

a peripheral

subunits,

of

the

Fl

sequence and

the

s subunits of

a,

b,

(5-8). all

organization

membrane portions N,N'-dicyclohexylF. in the order

FIFO of

of of

0006-291X/82/081257-08$01.00/0 1257

CopyrIght @ 1982 by Academic Press. Inc. All righls of reproducrion in any form reserved.

Vol. 105, No. 4, 1982

BIOCHEMICAL

the

genes

including

the

(6).

In

th e present

G and E on the

study,

genes

for

6 and c subunits

region

of

the

structure,

gene

physical

we determined

RESEARCH COMMUNICATIONS map of the

the

E. -__ coli sequence

nucleotide

genome of the

together with that of a possible terminator for F 1 Fo (pap operon). From the deduced primary

cluster

we

estimated and discussed

E subunits

AND BIOPHYSICAL

the possible secondary structure a possible nucleotide binding site

of the C and on the B sub-

unit. MATERIALS AND METHODS Preparation of plasmids and their fragments. Hybrid plasmids pFT1502 and pFT1503 were constructed in vitro by ligating a DNA fragment from hasn-5 -___ phage DNA (2) with a vector plasmid, pMCR561 (41. Portions of the E.Gli of genome carried by these plasmids are shown in Fig. 1. DNA fragm&ts portions of the gene cluster of FIFO (pap operon) (5) were prepared by digesting the plasmids with various restriction endonucleases. Determination of the nucleotide sequence. DNA fragments prepared by the 5'-end with sequencing strategy (Fig.1) were phosphorylated at the 32~~Y-ATP and Td-polynucleotide kinase. The nucleotide sequence was determined by the method of Maxam and Gilbert (1C). The restriction endonucleases and T4-polynucleotide Enzymes and reagents. kinase used in the present study were purchased from Takara Shuzo Co., Japan. All reagents used were of the highest grade available commercially.

RESULTS AND DISCUSSION Nucleotide

sequence

determined gene

the

for

the of

a

terminal

portion apart

sequence amino

sequence

of

Thus

the

for

the

45 base

pairs

from

Here

pair

long

DNA-segment

of

papB.

A termination

first

letter

from

agreed

6 subunit.

(7).

the

was deduced (Leu)

gene

(papB;

1855-base

from

acid

(11).

the

b subunit

sequence pairs

of

the

with

molecular

of

terminus the

adjacent TAA,

of the

the

appeared

the B subunit

1381

base acid

terminal

chemical was

amino

The amino

The carboxyl

by protein

we

nucleotide

to

codon.

sequence.

determined

weight

amino

initiation

nucleotide that

the

we determined codon,

of the

Previously

analysis

concluded

to

be

50,157. While

this

reported

the

paper nucleotide

dideoxynucleotide agreement

letter

(9).

of

the

with

and those

The amino from

the

termination

the protein

codon terminal

by protein

composition (Table

for

et -_

(atpD) __ results

the

of

E subunit.

al.

independently

determined were

by in

the

complete

The open reading

21 base pairs of

papB.

concluded

carboxyl

determined acid

papB sequencing

starts

(Ala-Met-Thr-Tyr-His-Leu-Asp) region

Saraste

of

Our

the gene

of 414 nucleotides of

preparation,

theirs.

sequence

comprised

in

sequence

method

with

Nucleotide

was

chemical

1). 1258

seven

to be present

residue the subunit

The

down-stream

(Met)

are

analysis determined

the last

amino

acid

residues

in the

amino

in complete of

frame

from

the agreed

terminal agreement

E subunit well

with

(11). that

vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-papD

HFE

14KaCbb

A

C

a

V

c ;

E

P ~~~~~~~~fl H

HHB

coding E

PB

H

PE

P

frame

P

DNA

E.coli

PSI I

Taql tipall Sau 3A tiinf

I

Alu

I

Hoc

Ill

Hha

I

I-1 pFT

1503

I

-._~

pFT

1502

I 5

L 0

I 10 bare

I 15

poirr

i 20

x 100

Fig. 1 Organization of the genes for F -F. and strategy of DNA sequencing. The direction of transcription of t +le gene cluster is shown at the top of the figure. The coding frame for each gene with its new and coding subunit is shown above the DNA. P nomenclature (pap) (5) indicates the promoter site of the pap operon. Cleavage sites of endonucleases are shown as follows:H, Hind111 ;B, BamHI ;P, =I, ;E, EcoRI. The cleavage maps with XI, HpaII, =A, fi1, HaeII, and -1 are -HinfI, also shown. Arrows indicate the sequenced DNA segments with the directions and approximate lengths. DNA fragments were prepared from plasmids pFT1502 and pFT1503, which cover the regions shown. The scale shown at the bottom corresponds to the numbers of nucleotide residues in Fig. 2.

We found the

that

sequence

one

base

data

reasons,

we

residue

(Met)

whereas

that

pair

reported

believe

by

that

deduced of

our

in

Saraste

et -2.

from

our

sequencing

analysis

than

theirs.

(9)

more

frequently our

than

the

weight Primary

in

data, 132 is

E.

we

14,914 and

sequence

homologous

with that

the

577)

and

(Table

that

the

TGA

al.

(9)

of

the

fi

structure that

tne

c1 subur,it

4 (7).

subunit as

binds

fCCD

at

amino

acid

sequence

1259

the

protein of

of

protein

and

terminal

Lys

and

TAA,

that

Ser

chemical is

its

used

them

(9). rather

molecular

E subunits.

has

a

The

sequence in

Glu around

data

residues,

indicated

a specific

following

by

138 and

in

chemical

contents

has

missing

carboxyl

determined

--et

(9).

is

codon,

E subunit

14,194

For

those

of

2)

The

A termination

instead

than

B subunit

The to

the Saraste

the

with

ii)

Fig.

(9).

coincided

iii)

by

of

al.

i)

not.

reported

indicates

in

reliable:

closer 1).

that

that

reported

did are

secondary

nucleotide

position

al

genes

possible

et

are

sequence

coli

rather

1793

Saraste

concluded

residues

position

data

cur

deduced

From

(C-G,

Fig.

residue the

Glu

partly 2.

It

(at

about

residue

is

is

Vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL Table -__

Amino

Acid

Amino

Composition

of

ACld

RESEARCH COMMUNICATIONS

1

the

i SubunIt

of

E.

Predicted from DNA Sequencr

coli

Fl

Reported'

males/14,914 Lys -xx His A.-g Asp + Asn Thr Ser ** Glu + Gin Pro GlY Ala Val Met Ile Len TY~ Phe

8 b 5 7 6 8 21 4 12 17 IO 5 11 12 4 2

9.3 6.4 5.4 7.7 5.8 8.4 20.9 4.2 12.3 17.0 a.4 4.7 9.3 11.8 3.9 2.2

* Reported mole per cent values for the I subunit (23) were multipled by 138 residues for the subunit. ** Hesldue numbers, estimated by Saraste et (91, for Lys ~- al. and Ser are 6 and 10, respectively.

similar

to

bacterium

those

The

possible by

that

both

B-sheet

the

beef

heart

in

mitochondria

We

to

looked

nucleotide

and

a thermophilic

is

(16)

and

present

to

that

of

whether

convergency study for

that

recA

of

of

enzymes

nucleotide sequence

sequence

between

initiation

of

is the

suggest a helix

known

and

to

bind

B subunit in

the

from @subunit

may

have

a in

kinase coli

enzymes

from

sites.

4).

it

fi sub-

It

is

muscle

not

enzymes would

and

for

the

skeletal

different

methods

of

porcine

However,

immunological

adenine

structure

sequence

(Fig.

in

bind

similar

the

(17)

that

be

compare

clear

at

indicate interesting

their

precise

binding. and papB

transcription A

in

E.

of

structure

sequence

sequences

binding by

in

a portion

of

similar

fold,

observed

deter-

results

structure

folding

adenylate

protein

nucleotide

these

that

were

The

site.

proteins

found

these

Flanking

region.

We

similar

This binding

homologous

these

binding.

unit

sites

a

E subunits 3)

Rossmann was

3).

f? and (Fig.

alternating

structure (Fig.

because

nucleotide

An

nucleotide

for

the (14)

a so-called

similar

the

of Fasman

spherical.

residues

related

and

a protein, A

240-330

structures Chou

are

(15).

be

of

subunits domains

about

nit

from

secondary method

nucleotide

to

Fls

(12,131.

mined

may

in

possible

a possible and

papG of

terminator is papG

terminator

The

sequence.

only

19 nucleotides

does

not

sequence

1260

take

long, place

was

found

in

intercistronic suggesting the around

that

intercistroposition

Vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

900

1600 1700 1800 1900

were part binding heart (Tyr) amino were Saraste found

previously determined (7). The amino acid sequence homologous with of the primary structure of the a subunit is underlined. A and B: DCCD site I" the 6 subunit of thermophilic bacterium PS3 F and beef mitochondrial F respectively (12, 13). C: A possible binding site of p-fluorosul -adenosine. +' onylbenzoyl-5Around this Tyr residue acid residues homologous with those reported for beef heart FL(20) observed. D: An amino acid sequence different from that determined by et al. (9). A sequence of Shine and Dalgarno (21) (underlined) was between papB and papG. __ ~

It

1840-1880.

between

the

region, mRNA,

as

should 5'-end

shown

because

Therefore, significance,

no the

be

noted

in

Fig.

5.

typical

as

the The

promotor

complementary such

that

of

moiety

a complementary mRNA

pap

of operon

structure structure

in

the

stabilizing

1261

pap may

structure operon be

is

and

the

transcribed

was

observed

in

the

mRiUA

mRNA

or

regulating

within could

have

observed terminator

as

a single

the

operon.

biological

termination.

We

Vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

f3 subunit

1 38

C subunit Fig. 3 Possible secondary structures of the Band E subunits. Possible secondary structure of the Band E subunits were determined by the method of Chou and Fasman (14). A possible nucleotide binding site ,a so-called Rossmann fold (15), was found at about residues 240-330 in the B subunit. The a helix contents of the B and E subunits were 27.2% and 43.4%, respectively. The B-sheet contents were 13.7% and 12.3%, respectively. The estimated content ofahelix in the ~subunit agreed well with data obtained by circular dichroism (23j.w ahelix,rVz/ B-sheet, 2 turn.

25 IIFVVGGPGSGKGTQC KVGLFGGAGVGKTVNM --145 160 265 10

A

a

C

-

04

D

GEGINFYGELVDLGVKEKLIE REGNDMHEMTDSNVID~VSL -- 185-

5'

285

GCUUUuUGAUGCUUGACU---l11111 I lllll

GG~JCUGAC~GAAAA

c ACGAA--------

‘tj 111:1l1111111 ACAGGC~GGCU~WJUIJIJ >8 1875

205

Fig. that

3'

4 A similar portion of the primary structure of the fi subunit to of enzymes capable of binding adenine nucleotide. A: Adenylate kinase from porcine skeletal muscle. B and D: 6 subunit of EF . C: recA protein of d omologous I_ E. coli. Amino acids are shown as single letters. amino acid residues are underlined. Number-s indicate residues in polypeptides from the amino terminal. Fig. 5 Possible secondary structure of mRNA of the -pap operon. The nucleotide sequences in 5'and 3'-end portions of putative mRNA of the pap 0peTYXl are shown. The nucleotide sequence of the promotor region and initiation of transcription in the operon were determined (24). site However, the 3'-end of mRNA in this figure is tentative. A complementary structure between the 5' -end moiety and terminator region of mRNA was found. A possible hair-pin loop structure (22) in the terminator region is also shown. Numbers of residues in the terminator region correspond to those in Fig. 2.

1262

BIOCHEMICAL

vol. 105, No. 4, 1982

AND BIOPHYSICAL Table

found

a

similar

complementary

E. (18). -~ coli Codon usage. of

the

Here

FIFO

components

used

reported closely that

in that

the

related

to

the

subunits

subunits

( y, subunits

f‘requency

of

the

optimal

OL, 13 and

c,

a and

FlFO

are

codon

Acknowledgements: unpublished results. Ministry of Education, Foundation and Toray

usage

in

c

of

a and

FlFC

the

optimal

gene

product,

is

codons,

defined 2).

codon

is

that

determined,

at

E.

G cells.

least

to

of

the the

some

(7). Ikemura Ikemura genes

different in

that

by

in

clearly

from suggesting

different

Recently,

in

protein,

operon

indifferentgenes

E subunits

b subunits),

each

tryptophan

usage

(Table

codon

subunits)

the

codon

optimal

the

of

amount of

(

the

for

mRNA

that

of

genes

frequency

6, of

of

than

frequency

the

frequency

abundant

the

other

the

in

previously

the all

2

sequence

reported

summarized

we

(19),

We

RESEARCH COMMUNICATIONS

is

We found in

the

less

abundant

amounts

of

all

by

the

extent,

gene.

We thank Dr. S.D. Dunn This work was supported Science and Culture Science Foundation.

for in of

allowing part by Japan, the

us to grants Yamada

cite from

his the

Science

REFERENCES

1) 2) 3) 4) 5J

Futai, M. and Kanazawa, H. (1980) Curr. Top. Bioenerg. " 181-215. Kanazawa, H., Miki, T., Tamura, F., Yura, T., and Futai, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76 1126-1130 Kanazawa, H., Tamura, F.. Mabuchi, K., Miki, T., and Futai, M. (19ROj Proc. Natl. Acad. Sci. U.S.A. 77 7005-7009 Tamura, F., Kanazawa, H., Tsuchiya, T., and Futai, M. (1981) FEBS Lett. 127 48-52 Kanazawa, H., Mabuchi, K., Kayano, T., Tamura, F., and Futai, M. (1981) Biochem. Biophys. Res. Commun. 100 219-225

1263

Vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Mabuchi, K., Kanazawa, H., Kayano, T., and Futai, M. (1981) Biochem. Biophys. Res. Commun. 102 172-179 Kanazawa, H., Kayano, T. Mabuchi, K., and Futai, M. (1981) Biochem. 7) Biophys. Res. Commun. 103 604-612 Kanazawa, H., Mabuchi, K., Kayano, T., Noumi, T., Sekiya, T., and 8) Futai, M. (1981) Biochem. Biophys. Res. Commun. 103 613-620 9) Saraste, M., Gay, N.J., Eberle, A., Runswick, M.J., and Walker, J.E. (1981) Nucleic Acids Res. 9 5287-5296 Maxam, A., and Gilbert, W.-(1977) Proc. Natl. Acad. U.S.A. 74 560-564 10) Communication. 11) Dunn, S.D. Personal M., Poser, J.W., Allison, W.S., and Esch, F.S. (1981) 12) Yoshida, J. Biol. Chem. 256 148-153 Esch, F.S., Bohlen, P., Otsuka, A.S., Yoshida, M., and Allison, W.A. 13) (1981) J. Biol. Chem. 256 9084-9089 Chou, P.Y., and Fasman, G.D. (1978) Advances in Enzymol. 47 45-148 14) 50 497-532 15) Rossmann, M.G., and Argos, P. (1981) Ann. Rev. Biochem. A., Muller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, 16) Heil, I ., and von Zaber, I. (1974) Eur. J. Biochem. 43 131-144 Horii, T., Ogawa, T., and Ogawa, H. (1980) Proc. Natl. Acad. Sci. 17) U.S.A. (1980) 77 313-317 C.,?latt, T., Crawford, I.P., Nichols, B.S.,M Chrisie., 18) Yanofsky, Van Cleemput, M., and Wu, A.M. (1981) Nucleic Acid G.E., Horowitz, Res. 9 6647-6668 T. (1981) J. Mol. Biol. 151 389-409 19) Ikemura, 20) Esch, F.S., and Allison, W.S. (19% J.Biol.Chem. 253 6100-6106 L. (1974) Proc. Natl. Acad. Sci. U.S.A. 71 21) Shine, J., and Dalgarno, 1342-1346 M., and Court, D. (1979) Ann. Rev. Genet. 13 319-353. 22) Rosenberg, P.C. and Smith, J.B. (1980) Biochemistry 19 526-531 23) Sternweis, K., and Futai, M. Submitted for publication. 24) Kanazawa, H., Mabuchi, 6)

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