Nucleotide sequence specificities of guanylate residue-specific tRNA methylases from rat liver

Vol. 40, No. 2, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NUCLEOTIDE SEQUENCE SPECIFICITIES OF GUANYLATE RESIDUE-SPECIFIC Yoshiyuki Biology

Received

specifically

the

in

for

examples),

purified

problem that

liver

the

in

catalyzes and tRNASer.

tRNAPhe.

The other

methylguanosine of

RNase Tl

or pancreatic

the

catalyzes

methylation

stem and the located

since

Institute,

yet

since

tRNA's

with

of

a specific

nucleotide

structure

the

is

(See

methylated

unfractionated

are

recognition

review

bases

tRNA was used

in our

methyl

tRNAfMet, tRNAzer

groups

as a

This

specific

methylases

One of

into

g.

coli in

and tRhAyU,

methylase

the

located catalyzes

former

between methylation

and the

tRNAPhe, and

produces major

14C-methylated

that

dihydrouridine-stem

the

in

methylases

tRNAIal,

is

individual

paper

tRNAyal

and also

E2-dimethylguanosine

residue

these

product

tRNAger from

so we could

precisely,

sole

demonstrated

guanylate

The other

on tRNA.

laboratory

was the where

derived

more

of guanylate sites

of

RNase clearly the

available

two species

oligonucleotides

the

methylated

clover-leaf

by tRNA methylase

least

methylates

between

the

recognize

experiments,

their

of

a particular

the positions

known,

incorporation

CCA-stem.

tRNA methylases

as in

However,

_N2-Methylguanosine

except

Analyses

that

same region

recognition

are at

differing

tRNAMet

suggested

E_. coli

of

there

exclusively

residue

Cancer Center Research Chuo-ku, Tokyo, Japan

seems probable

are not --in vitro acceptor in previous

reports rat

and Susumu Nishimura

National S-chome,

have

located (4)

Numerous study

(l-3) This

tRNA.

by Zachau methyl

Division, Tsukiji

studies

of

introduced

Kuchinov

June 8, 1970

Recent region

tRNA METHYLASES FROM RAT LIVER

ES-

product.

tRNA's

by

methylase

the

dihydrouridineof

the

guanylate

anticodon-stem.

MATERIALS AND METHODS Amino acid specific tRNA's from E. coli B were obtained by column chromatographies on DEAE-Sephadex A-50 (5), followed by benzoylated DEAE-cellulose (6) or/and reverse phase partition column chromatography (7). Details of the procedures were described previously (5,8-10) or will be published elsewhere. The tRNA's used were at least 70 to 95 % pure, judging from the capacities to accept individual amino acids and the chromatographic profiles of their digests with RNase Tl. Unfractionated methyl deficient tRNA was prepared by the method of Zubay (11) from E. coli KlZ-58-161 RCrel which was grown in a minimal medium containing 0.0003 * On leave Fukuoka,

from Cancer Japan

Research

Institute,

Faculty

306

of Medicine,

Kyushu

University,

%

Vol. 40, No. 2, 1970

L-methionine. modification. Nl-methylguanosine -

BIOCHEMICAL

Rat liver Authentic were

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

tRNA was prepared as described by Zubay (11) with samples of N2-methylguanosine, N_2-dimethylguanosine of our Institute. kindly supplred by Dr. M. Saneyoshi

a slight and

Eight g of liver Preparation of guanylate-specific methylase from rat liver: from adult female rats were homogenized with four volumes of cold 0.25 M sucroseThe homogenate 0.01 M Tris-HCl (pH 8.Oj-O.01 M MgC12-0.001 M p-mercaptoethanol. The crude extract thus obtained (25 ml) was centrifuged at 105,000 x g for 1 hr. was applied to a column of hydroxylapatite (BIO-GEL HTP; column size: 1.5 cm x 20 cm), and washed with 150 ml of 0.01 M Tris-HCl (pH 8.0)-0.001 M EDTA (pH 8.0)Elution was achieved with a linear gradient 0.001 M p-mercaptoethanol (buffer A). obtained by placing 200 ml of buffer A in the mixing chamber and 200 ml of 0.01 M Tris-HCl (pH S.O>-0.001 M EDTA (pH 8.01-0.001 M p- mercaptoethanol-0.05 M potassium phosphate buffer (pH 8.0) (buffer B) in the reservoir. Solid ammonium sulfate was added to 60 % saturation to the eluate containing methylase activity, The precipitate was dissolved in a minimal volume of buffer A, and dialyzed against buffer A, Assay of tRNA methylase activity: The procedure was essentially The reaction mixture contained 10 kmoles of by Tsutsui --et al. (12). (pH 8.0), 1 pmole of MgC12, 1 pmole of reduced glutathione, 4 wmoles labeled S-adenosylmethionine (specific activity: 20 C/M), 0.05 0.D. and an appropriate amount of methylase in a final volume of 0.1 ml. bation at 37OC for 1 hr, 0.05 ml of the reaction mixture was applied paper disk. Disks were washed three times with cold 5 % trichloroacetic then successively with ethanol-ether mixture (l:l, v/v) and ether. was counted in a liquid scintillation counter. Large scale preparation of methylated tRNA in vitro: mixture described above were treated with an equal volume precipitated from the aqueous layer by adding 0.1 volume of the respective tRNA and 2.5 volumes of cold ethanol. minimal volume of 0.01 M Tris-HCl (pH 7.51-0.06 M KCl-0.001 dialyzed first against the same buffer, and then against

4

-I

that described Tris-HCl of l4C-methy1, units of tRNA After incuto a filter acid and Radioactivity

Five ml of the reaction of 88 % phenol, tRNA was of 5 M NaCl, 10 0.D. units tRNA was dissolved in a M p-mercaptoethanol and distilled water.

n -

20

2 7

0

;

IO fj

80

40

FRACTION Fractionation Fig. 1. chromatography. -,

of rat liver UV absorbance;

1213

NUMBER tRNA methylase by hydroxylapatite -O-O- , radioactivity.

column

BIOCHEMICAL

Vol. 40, No. 2, 1970

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS As shown in by hydroxylapatite E. coli acid

column

methyl

act

were

isolated

tested.

acceptors

for

tRNAfMet, while

these of

methylate

tRNAfMet,

tRNAser

methylases from

inert

I and II

methylases

I and II

To characterize

Table liver

I. Methyl methylases in -Extent

the

of

tRNA?l,

tRNA?l,

tRNAPhe,

tRNAieu.

both

rather with

acceptor vitro

Table

to its of

the

capacities

of methylation

I also These

shows

recognition

methylated

of

(cpm/O.05 I

ml of

Methylase

II

but

could

it

tRNAASp,

formed

tRNA's

on methyl-

with

150

440

530 2340

140

1790 4580

4110

210

Methylase

tRNAyt

74

110

510

33

240

770

Yeast

tRNA

32

220

520

2380

3270

110

260

480

213

64

350

710

1440

1280

460

1730

1600

540

84

34

240

70

4.5 230

Each reaction mixture contained numbers listed are averages of values using different enzyme preparations.

0.7 mg of obtained

308

III

170

tRNAPhe

tRNA

rat

mixture)

290

liver

tRNAzlU differs

210

Rat

not

III

tRNAAsP

Methyl deficient g. coli tRNA

poor

that

Methylase

tRNA

of

catalyzed

indicate

tRNA

E_. coli

very III

on tRNA.

E, coli reaction

were

methylase

nucleotides

individual

to type

methylated

methylase that

three

abilities

clearly

site

amino

these

extensively

that

using

on the

and tRNAfer

results but

for

depended

contrary,

properties,

respect

structure

tRNAMet

species

various

had different

tRNAMet and tRNAzer

methylases, similar

tRNA's methylation

On the

of

as acceptors

and tRNAkeU were

tRNAPhe,

or

with have

these

two methylases.

methylation were

that extent

three

was measured

The abilities

B to serve

tRN4zer

into

activity

acceptor.

I shows the

was separated

Methylase

E_. coli

and that

extensive and tRNA:y'

from

Table

acceptors,

For example, methylase. by methylases I and II, methyl

tRNA methylase

tRNA as methyl

tRNA's

as methyl

liver

chromatography.

deficient

specific

methylases

1, rat

Fig.

the enzyme preparation. The from two separate experiments

well

BIOCHEMICAL

Vol. 40, No. 2, 1970

ation

of each

tRNA,

14C-methylated

14C-methylated

tRNApl

activities indicating

that

determine

the

treated

with

sides

were

previously

chromatography

were

close

in all

cases

coli

of

(91,

together the the

alkaline

the

was the only -N2-methylguanosine z2-dimethylguanosine tRNAPhe, while not

shown in

from

the

tRNAkeU,

figure,

it

bases,

N_2-methylguanosine

methyl

acceptor.

that

catalyzed

resulting

of

To

tRNA was

in Fig.

tRNAp1

tRNAger, was the

of two different

depending

nucleo-

PJ2-dimethylguanosine As shown

tRNAfMet, from

formation

radio-

as marker,

14C-methylated

from

product

and N_2-dimethylguanosine,

N 0

the

used

by

was methylated.

N2-methylguanosine the

All

the RNase T2 digest

was the major

and

analyzed

(13).

tRNA's

product

II,

hydrolyzed

was first

N2-methylguanosine, and Mandel (14).

14C-methylated

II

in

and the

by Hacker

methylase completely

3'phosphate

residue base,

markers

was shown

Thus methylase

digest

guanosine

guanylate

with

with were

by Tada et al.

with

methylated

as described

III

and each

phosphatase,

chromatographed

and N1-methylguanosine

and tRNAger

methylase

as described

eluted structure

2.

tRNAfMet

and tRNAPhe with

by RNase T2 as described Dowex 1 column

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

upon the

2,

and

Although major

product methylated

tRNA used

as

1

X2 Fi

;' -~

H

-l--l-

tRNA”;’

I

I

I

I -J-

0

10

20

3( 10

DISTANCE

Fig.

Characterization

2,

To determine tRNA's,

tRNAfMet,

primary

sequences

excess pancreatic

of

the

the

exact

of

l4Cmethylated

ORIGIN

I

20

:0

(cm)

nucleoside

by paper

chromatography.

location

of the nucleotides methylated in individual were used as methyl acceptors, since their tRNAVal and tRNARhe 1 The 14C-methylated tRNA's were mixed with are known (X-18).

corresponding control Each RNase digest

RNase.

from

I

10

an

either with RNase Tl or tRNA, and hydrolyzed was fractionated by DFAE-Sephadex A-25 column

Vol. 40, No. 2, 1970

chromatography

in the

RNase Tl digest were

eluted

eluted (Fig.

of

in

(Fig.

was only

In

the

in

the

From these

concluded

that of

digest

nucleotide

the

g,

coli

digest

of

of

the

the

GpCp,

RNase Tl

phosphatase,

digest

v/v/v)

as solvent.

CpUpCpG,

indicating

that

the

methylated

show unambiguously

that

the

site

of

radioactivity

guanylate To determine

was

the

which

RNase

oligofrom

the

by treatment

markers

using

n-propanol-

coincided

oligonucleotide of

in

it

obtained

was dephosphorylated with

eluted

tRNAfMet,

oligonucleotide

methylation

and pCpGp were

RNase,

The radioactivity

of

the

GpUp and ApUp were

structure

and chromatographed

In

radioactivities

CpUpCpGp

pancreatic

radioactive

(9).

the

14C-methylated

(35:10:35,

results

with

known primary

NH40H-HZ0

marker

all

pCpm2Gp or CpUpCpm2Gp.

methylated,

fraction

II,

CpCpUpGp,

where

containing

was either

alkaline

where

region

and the

previously

methylase

tRNAfMet

dinucleotide

RESEARCH COMMUNICATIONS

as described

with

region

oligonucleotide

was actually

tetranucleotide

of 7 M urea,

results

tRNAfMet

AND BIOPHYSICAL

methylated

tetranucleotide

3a).

Tl

cont.

presence

tRNAfMet

the

detected

3b).

with

BIOCHEMICAL

with

was CpUpCpm2G. tRNAfMet

the These

by methylase

1.0

20

0.5

10

2 s 4

II

z ‘a

2 ” 1.0

20 IO

0.5

100

50

200 FRACTION

100

NUMBER

DEAE-Sephadex A-25 column chromatography of RNase digests of 14CFig. 3. methylated 2, coli tRNA's. a) RNase Tl digest of tRNAfMet, b) pancreatic RNase digest of tRNA=, c) RNase Tl digest of tRNA pl,,d) pancreatic RNase digest of tRNAya1; -, UV absorbance: -O-O-, radioactivity; column size, 0.3 x 150 cm; Gradient elution was achieved by placing 200 ml of 0.02 M Tris-HCl (pH 7.5j-O.14 M NaCl-7 M urea in the mixing chamber, and 200 ml of 0.02 M Tris-HCl (pH 7.5)0.7 M NaCl-7 M urea in the reservoir, Each fraction contained 2 ml of effluent.

was the the

guanylate

residue

dihydrouridine-stem the Similarly,

site

from analysis of digests In the RNase Tl digest,

at

the

27th

position

from

the

5'QH

and the anticodon-stem (Fig. 4). of methylation of tRNAlVal by methylase of the

l4C-methylated radioactivity

tRNAyal was only 310

end, III

located

between

was determined

with RNase Tl or pancreatic RNase. present in the fractions contain-

BIOCHEMICAL

Vol. 40, No. 2, 1970

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AOH C

AOH E

i pk.; E-E &4 &,,G*CCGG G cCGACG . . AG . .

U G&AGC

Yi pG . c g:E 8:;

CC”**

@Jc GG

t

;CG*

TYJC

U m’G

UCG fg.$*G “G:: .

GG

,&&A G

tRNAVa’1

ing

Clover-leaf methylation.

of

ApUpCpCpCpGp

radioactivity

was

GpApUp

and

the

a similar exclusively end,

at

methyl

which

was

the

ated

known

yet.

present

not

Analysis in

the

of

methylation

from

the

5 ‘-OH

end,

same

site with

as

at

that

II the

not

shown

in

showed

that

RNases

showed

position

the region the

figure,

tRNAPhe

was

from

location

structure that

GpGpCp,

the

5’-OH

tRNApl.

exact

primary

the

clearly the

in

the

digest,

in

10th

methylated

methylase

with

the

.ting

ApGpCp, was

Although

since

digests

RNase

namely

tRNARhe

residue

indica

containing

site

guanylate

tRNAyal,

pancreatic

the

14C-methylated

determined,

of

Cpm$GpC

the

dihydrouridine-stem.

tRNAser

and (27).

fraction

position

the

been

In

trinucleotide

using

the of

could

the

tRNAfMet acid

3~).

Therefore

10th

at

exactly

On methylation guanosine

the

experiment

of

(Fig.

3d).

em and

CCA-st type

in

(Fig.

residue

between

ApUps4UpApGp

eluted

GpGpTp

guanylate

structure of E_, coli V, uridin-5-oxyacetic

and

TvJC

C U mAt VAC

tRNAfMet 4. site

G

i;CCG cU C * G G m7G C-G G

CUCG*

C”, ‘A u c A”*

Fig. the

c”A

$&“GCC

A

of

N2-dimethylthis tRNA is

of

E2-dimethylguanosine

was

sequence.

DISCUSSION Methylase position

in

III

catalyzed

both

methylguanosine, in site which ated

these

The tRNA’s

specifically contains by

coli

E.

methylase

is

methylation

of

and

tRNAPhe

tRNApl common

nucleotide

S4UAGCUCAG, recognized

s4UAGCUCAG III

also

in

the supports

the

guanylate

resulting

sequence

suggesting by

the

that

around this

methylase,

same

region the

above

residue in

the the

ref.

conclusion,

E, was It

coli

N2-

guanylate is

the

tRNAMet

exclusively is

10th of

sequence that

19)

the

methylated

nucleotide

Evidence (See

at formation

interesting

methylto

not

Vol. 40, No. 2, 1970

note

that

wheat

the

germ

UAm2GC sequence

tRNA*he

AGC, by itself since

the

E. & It

BIOCHEMICAL

(2$),

is

not

guanylate

also

from

the

5'-OH

methylase

that end is

III,

was not

since

methylated

recognizes residue

not

sufficient

for

residue

II

methylated

residue

yielding

Dimethylguanosine pyrimidine,

This

of

located were

of

These

of

a nucleotide

as in

terms

of

the

at the

27th

formed

in

result

agree

is

tRNA2er

a pyrimidine-m$G-pyrimidine

found

in

the

that

the

with

sequence

fact

at

of

in several

residue

and tRNA*he

in a CGU sequence

was not

methylated

formed

by methylase

Namely

N2-methylguanosine

guanosine a single

sequence,

from

the

However,

the

case of

synthesis Further

and their

depending

was formed formed

participates it yeast

in is

is

yeast

from

possible

in which

fragments

on the as methyl

from

14C-methylated of both

that

methylase

different

acceptors

tRNAfMet, tRNAger,

N2-methylguanosine II is a mixture

tRNA methylases

and fi2-dimethylguanosine

recognition

region

14C-methylated

of

tRNA methylase

are

in progress,

by the

occupied

by

since a in E. & tRNAp1 product

acceptor, N2-dimethyl-

is

probable that and N2-dimethylof two enzymes,

seem to be involved respectively using

(See

sequence

while It

germ

II,

The methylation tRNA used as methyl

the

molecule.

and wheat

I and II, upon

formation

of z2-methylguanosine studies

in a different

by methylases

differed

was predominantly enzyme

guanosine, as in

II

located

the

guanylate

N2pyrimidine-G-

ref. 20-26). As in the case of methylase III, the pyrimidine-G-pyrimidine is not the sole determining factor for the recognition of methylase guanylate

the

around

same region

tRNA's

methylase

was methylated

the

by

position

the

around

sequence

III.

position

residue this

pyrimidine-G-pyrimidine.

was also

10th

guanylate

structure

The nucleotide CGU, namely

of

by methylase

sequence

in tRNAfMet

III,

region

show that

secondary

position

sequence,

at the

residue

results

terms

tRNAfMet

this

(20),

by methylase

methylated residue

a guanylate

III.

tRNAPhe

in a different

not

methylation

contains

yeast

recognition

a guanylate

N2-methylguanosine,

guanylate

the

in

as well

methylated, The guanylate

which

by methylase

of

The trinucleotide

(22,23),

and tRNAzyr of only

RESEARCH COMMUNICATIONS

same region

the AGC sequence

tRNA*he

all

the

determining

presence

tRNAfMet at

tRNATyr

factor in

in

the

a guanylate

methylase

sole

tRNAp1,

clear

present

and yeast the

residues

tRNAfMet,

is

is

AND BIOPHYSICAL

purified

in

(27). tRNA's

REFERENCES 1.

2. 3. 4. 5.

6. 7.

8.

B. C, Baguley and M, Staehelin, Biochemistry 7, 45 (1969) 8, C. Baguley and M. Staehelin, Biochemistry 8, 257 (1969) D. G. Streeter and B, G. Lane, Biochim, Biophys. Acta, 199, 394 (1970) H. G. Zachau, Angewande Chemie, International Ed., 8, 711 (1969) S. Nishimura, F. Harada, U. Narushima and T. Seno, Biochim. Biophys. Acta, 142, 133 (1967) I. Gillam, S. Millward, D. Blew, M. von Tigerstrom, E. Wimmer and G. M. Tener, Biochemistry, 6, 3043 (1967) A. D. Kelmers, G. D, Novelli and M. P, Stulberg, J. Biol. Chem., 240, 3979 (1965) H. Ishikura and S. Nishimura, Biochim, Biophys. Acta, 155, 72 (1968) 312

Vol. 40, No. 2, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T. Seno, M, Kobayashi and S. Nishimura, Biochim, Biophys. Acta, 169, 80 (1968) K. Oda, F. Kimura, F. Harada and S, Nishimura, Biochim. Biophys. Acta, 179, 97 (1969) 4, 347 (1962) 11. G. Zubay, J. Mol. Biol., P. R. Srinivasan and E. Boreck, Proc. Natl. Acad. Sci., U.S., 56, 12. E, Tsutsui, 1003 (1966) M, Tada, M. Tada and K. Yagi, J. Biochem. (Tokyo), 55, 136 (1964) 13. B, Hacker and L, R, Mandel, Biochim. Biophys. Acta, 190, 38 (1969) 14, B, F. C, Clark and S, Cory, Nature, 218, 232 (1968) 15 * S, K. Dube, K, A. Marcker, Nature, 222, 278 (1969) 16. M, Yaniv and B. G, Barrell, F, Harada, F. Kimura and S. Nishimura, Biochim, Biophys. Acta, 195, 590 (1969) 17, B, G, Barrel1 and F. Sanger, FEBS Letters, 3, 275 (1969) 18. S, Cory, K, A, Marcker, S, K. Dube and B. F. C. Clark, Nature, 220, 1039 (1968) 19. S. H. Chang, A, Stuart, R. D. Faulkner, R. M, Hoskinson and 20. U, L, RajBhandary, H, G, Khorana, Proc. Natl. Acad. Sci,, U.S., 57, 751 (1967) Proc. Natl. Acad. Sci., 21. B, S, Dudock, G. Katz, E, K. Taylor and R. W. Holley, U.S., 62, 941 (1969) and H, Kung, Science, 153, 531 (1966) 22 * J. T, Madison, G, A, Everett S, Hashimoto, M, Miyazaki and S. Takemura, J. Biochem. (Tokyo), 65, 659 (1969) 23, H, G, Zachau, D. DUtting and H. Feldmann, Hoppe-Seylers 2. Physiol. Chem., 24. 347, 212 (1966) R, W, Holley, J. Apgar, G. A. Everett, J. T. Madison, M. Marquisee, S, H, 25. Merrill, J, R, Penswick and A, Zamir, Science, 147, 1462 (1965) M. Murakani and M, Miyazaki, J. Biochem. (Tokyo), 65, 489 (1969) 26. S. Takemura, J, H, Phillips and K. Kjellin-Straby, J. Mol. Biol., 26, 509 (1967) 27. F. Harada and S, Nishimura, Biochem. Biophys. Res, 28 * K, Murao, M, Saneyoshi, Commun., 38, 657 (1970)

9. 10.