Affinity chromatography of aminoacyl-tRNA synthetases on specific tRNA columns without prior purification of tRNA

Vol. 60, No. 4, 1974

BIOCHEMICAL

AFFINITY CHROMATOGRAPHY SPECIFIC tRNA COLUMNS Catherine

September

RESEARCH COMMUNICATIONS

OF AMINOACYL-tRNA SYNTHETASES ON WITHOUT PRIOR PURIFICATION OF tRNA

M. Joyce and Jeremy

The Dyson Perrins Laboratory,

Received

AND BIOPHYSICAL

University

R. Knowles*

of Oxford,

Oxford

OX1 3QY,

U.K.

3,l.974

SUMMARY. A method is described for the purification of aminoacyl-tRNA synthetases by chromatography, using a column of tRNA lacking the cognate tRNA, followed by a column of the cognate tRNA. The ability of the enzyme to discriminate between cognate and non-cognate tRNA is exploited in a novel and rapid preparation of the two columns. The purification graphic

techniques

enzymes

is usually

tiresome,

in crude cell extracts.

particularly

helpful

So far, affinity natural

of aminoacyl-tRNA

synthetases largely

In such a situation

for synthetase

amino acids (l-3)

and tRNA

tRNA have proved

more satisfactory.

However

column

of pure tRNA cognate purification

have pointed

out that such methods are unable

recognises

general

features

affinity

columns:

lacking

the cognate

Y

Copyright All rights

0 1974

features

of all tRNA’s.

Present address:

Abbreviations:

one might expect

specific

a column tRNA,

(4-8);

of a single

to separate tRNA,

this latter

made use of a

(4,5,7)

did not achieve

Ebel and coworkers

the desired

a method

preceded

Harvard

University,

recognising

involving

two of tRNA

proteins.

Cambridge,

Phe-RS, phenylalan I-tRNA synthetase; similarly for other see Scheme. synthetases. tRNA &al , tRNAox, tRNAoxmed:

1278

(6)

synthetase,

by a column

to remove other tRNA-binding

Department of Chemistry, Mass.02138.

the use of the

from other enzymes

proposed

tRNA,

time.

of these the methods involving

those methods which

They therefore

should be

in a shorter

have involved

from this technique.

of pure cognate

by Academic Press, Inc. in any form reserved.

of reproduction

isolation

of these

chromatography

with the target enzyme

the dramatic

which

affinity

factors may be obtained

substrates,

single

chromato-

due to the low concentration

since larger purification

methods reported

by conventional

In this

Vol. 60, No. 4, 1974

BIOCHEMICAL

way yeast Phe-RS

was purified

Ser-RS to 8590%

purity

of the necessary

from non-cognate

this very distinction with tRNA; avoiding

to homogeneity

tRNA.

(between

it is possible

a laborious

(6) and more recently

A particular

cognate

to exploit

fractionation

pair of columns

requires

and non-cognate

the desired

of tRNA by physical

techniques.

the actual

sequence

achieving

in this way a considerable

advantage

of this system is that a pure enzyme preparation

specificity

can be achieved

synthetases

and crude

of reactions

cognate

tRNA,

susceptible

to oxidative

may be coupled separation

tRNA.

affinity

cleavage

so a borohydride

in subsequent

the cognate

tRNA susceptible,

thus providing

reactions.)

to differentiate

precedent;

several

(see e.g.:

11,12).

tRNA,

reduction

of crude

The resulting

of the and dialdehyde

48, thus achieving

both the

of one of the non-cognate out to prevent

of the aminoacyl

tRNA its

group leaves

and coupling

to the carrier

column.

aminoacylation

between

(9).

of the oxidised

to oxidation

since total

remains intact

(10) is carried

Hydrolysis

in its turn,

A further

out using unfractionated

and the production

removal

the second affinity

The use of the enzymic oxidation

of Sepharose

(In practice,

participation

matrix,

by sodium periodate

material,

at the 3’-terminus

tRNA this feature

from non-cognate

to the column

amino acid to a mixture

masks the -cis-glycol

derivative

tRNA during

is not required

is carried

thus

We have used

in both time and effort.

a single

in the non-cognate

separation,

and non-cognate

is coupled

When the reaction

columns.

may not be complete,

cognate

tRNA

saving

by adding

ta a hydrazide

of cognate

necessary

in which

temporarily

while

between

makes

in its interaction

this fact to effect

to discriminate

the separation

synthetase

species)

reaction

aminoacylation

rat liver

solely

aminoacyl-tRNA

the enzyme

tRNA,

RESEARCH COMMUNICATIONS

(8).

The preparation of cognate

AND BIOPHYSICAL

cognate

reaction

in conjunction

and non-cognate

methods for the purification

1279

tRNA

with periodate

is not without

of tRNA are based on this principle

Vol.

60,

No.

4, 1974

BIOCHEMICAL

AND

Mixed

a:

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

tRNA

charging

+RNA””

tRNA-Va’

b OA

(all

(Val-tRNAva’) Hd

6-Val

HO

b:

OH

oxidation

1

tRNAVa’ M

tRNA-“*’

+ HO

(tRNA-Val) ox

O-V&l

c:

tRNAVa’

coup1

ing

-

I

+

+ any HO

other

uncoupled

tRNAox

-Val

O-&II

$

separation

of

Sepharose I

e:

reduction

tRNA-Val

tRNAVa’

+ HO

(tRNA;;f;ed) HO

O-&l

OH

I

Scheme.

f:

discharging

g:

oxidation

Preparation

of and

of affinity

Val-tRNAVa’ coupling

and

1280

of tRNAVa’

anti-affinity

columns

tRNAs)

BIOCHEMICAL

Vol. 60, No. 4,1974

RESULTS AND

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION

tRNA columns designed

for the purification

of Val-RS

were prepared

as shown

in the Scheme. When an impure enzyme column, higher

the elution capacity

However,

profile

gel electrophoresis

of tRNAVa’

Improved mixture

material Val-RS

purification

was achieved on a column

and concentrated;

relative

then chromatographed

on the tRNAVat

-Val

a single

protein

proteins.

the crude enzyme sample was applied

which

frontally.

A suitable (Fig.2);

column

: the enzyme

assays showed,

column

with other synthetases.

above)

by first subjecting

to other synthetases.

the much

from other tRNA-binding

of tRNA

Val

showed three major components.

since (as mentioned Val-RS

on the tRNA

demonstrating

compared

fractions

washed to remove non-bound

was pooled activity

for Val-RS

of the pooled

of separating

to chromatography

and the column

material

to be expected

*IS incapable

was chromatographed

shown in Fig. 1 was obtained,

of this column

This result was entirely

solution

eluted

This

as expected,

an increase

quantity

of this pool was

gel electrophoresis

of the

a The aminoacylation mixture contained: 104mM-trien-HCl buffer, pH 7.2, -%g(OAc)2 (IO,4 mM), KCI (10.4 mM), ATP (2.1 m/v!), valine (0.83 mM), crude E .Coli tRNA (1.67 mg/ml) (Wh a t man Biochemicals Maidstone, Kent, U.K.), and Val-RS (100-1000 /,runits/ml). After 2 h at 4 d C, the reaction was quenched by cooling in ice and the tRNA recovered by phenol extraction followed by ethanol precipitation. b tRNA was oxidised according to Remy et al. (6). c Qxidised tRNA was coupled to the carfiatrix (6) [commercial CNBrictivated Sepharose (Pharmacia, Uppsala, Sweden) derivatised, in accordance with the manufacturers’ instructions, with adipic acid dihydrazide (13).1. d Functionalised Sepharose was recovered by filtration, washed and treated with NaBH4 (6). The tRNA-containing washings were combined and the tRNA recovered by lyophilisation for use in the following stages. e Reduction of any residual oxidised tRNA was carried out using excess NaBH4 p r&mg tRNA) in lM-phosphate buffer, pH 7, for 2 h at room temperature in the dark. f The tRNA was dialysed against 0.1 M-glycine buffer, pH 10.3, for 5 h at Gem temperature. Typically the fiq:folumn comprised 6 ml (settled bed volume) Sepharose carrying and the second column 1 ml Sepharose carrying 1 r&ml ,$$leNA ’ .

1281

in

Vol. 60, No. 4, 1974

BIOCHEMICAL

cp.m in

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

c.pm in assay

assuy

0

Figures.

Elution

profiles

of tRNA columns

(for details

see text)

All columns were run using O.OSM-acetate buffer, pH 5.5, containing glycerol (10% v/v), bCl (IOmM), EDTA (0.1 mM), and P-mercaptoethanol (20mM). The enzyme samp Pe was a partially purified mixture containing Val-, Leu-, Met-, and Tyr-tRNA synthetases from B.Stearothermophilus (kind gift of Drs. K. Sargeant and A. Atkinson, Microbiological Research Establishment, Porton Down, Salisbury, Wilts., U.K.). After extensive washing to remove non-bound protein, the bound protein was eluted with a linear gradient (at c ) of KCI (O-1M). Fractions were assayed for Val-RS ( -t ), Leu-RS (+), and Tyr-RS ( 4) as described by Wilkinson and Knowles (14): A280 (- -. - . . e). Fractions marked w were pooled and concentrated.

fractions

containing

Val-RS

In principle, general provided

should

the separation

tRNA features

we achieved

not bind - however,

ASSESSMENT

AND

now showed a single of Val-RS

can be accomplished

that one column

For example,

activity

is capable a similar

from other enzymes which using any pair of specific

of retaining

Val-RS

result using a tRNA

see below),

major component.

followed

while Leu

by a tRNA

recognise

tRNA columns,

the other is not.

column Val

(to which Val-RS

column.

SCOPE OF THE METHOD

In this paper we have described

the successful 1282

development

and application

Vol.

60,

No.

of a method chromatography drawbacks

BIOCHEMICAL

4, 1974

for

the

purification

on specific of affinity

AND

BIOPHYSKAL

of an aminoacyl tRNA

columns.

chromatography,

tRNA Our

notably

synthetase

method the

RESEARCH

time

c.p.m. in assay

avoids and

effort

COMMUNICATIONS

using one

of the

main

expended

in

c.p.m. in assay

16000 ,-

8coo

c.p.m. in assay

0

5

IO Fraction

1283

15 Number

20

25

affinity

the

Vol. 60, No. 4, 1974

preparation

BIOCHEMICAL

of the ligand

to the affinity

matrix. concurrently

preparation

barely

enzyme,

(in this case, the purified Since the separation

takes place

to be coupled

from non-cognate

tRNA

for the two coupling

reactions.

complete

of the target

described

did not effect still

being detectable

the reactions

upon which

depends

(in particular

the charging

and this results in some overlap

the separation reaction

degree

that,

purification

the entire

on gel electrophoresis. of cognate

This is

from non-cognate

tRNA

to completion,

of the two columns.

Nevertheless,

of purification

with further

column,

(15)) may not proceed

in specificity

that a very substantial

is reason to believe

of cognate

tRNA)

the time required

because

there

cognate

of the first affinity

minor contaminants

it is clear

RESEARCH COMMUNICATIONS

with the preparation

exceeds

The columns

AND BIOPHYSICAL

refinement

has been achieved, of the method,

and

this could

be

improved. Application be possible,

of this method

subject

to the following

First, the desired

problem

but unable

in attempting

both Val-RS

heterologous similar

amino

Secondly,

one from the other. of Leu-RS: with,

e.g.,

In this situation, satisfactory separation

mischarging

of co-purifying

We encountered

the tRNALeU Tyr-RS)

errors are more frequent,

for the successful

should also

tRNA;

capable

tRNA Val by B.Stearothermophilus

acids are concerned. should permit

only its cognate

a pair of columns

(compared

of E.Coli

system, charging

the same organism

*

to separate

and Leu-RS strongly

to be due to mischarging

must charge

produce

the purification

synthetases

conditions.

synthetase

of a second tRNA species would two synthetases

to other aminoacyl-tRNA

particularly

column

this

retained

(Fig.3).

This seems

Leu-RS. where

In a

chemically

use of enzyme and tRNA

application of cognate

,

from

of our method.* from non-cognate

tRNA,

Val As mentioned above, this tRNALeU column was used in conjunction with a tRNA column in the purification of Val-RS; in this case the ability of the tRNALeU column to bind Val-RS merely lowered the yield rather than affected the purification.

1284

BIOCHEMICAL

Vol. 60, No. 4,1974

it is imperative reaction. which

that the cognate

hours (16),

it seems likely

We are grateful

RESEARCH COMMUNICATIONS

tRNA remains aminoacylated

Since this takes place most aminoacyl-tRNA’s

AND BIOPHYSICAL

at pH 5 and room temperature,

have a half-life, that this condition

to the Science

with respect will

invariably

Research Council

during

the oxidation

conditions to hydrolysis,

under of several

be met.

for support.

REFERENCES 1. Beikirch H., von de Hoar F. and Cramer F,. (1972) Eur.J.Biochem. 26, 182-190. 2. Robert-Gero M. and Waller J-P. (1972) Eur. J. Biochem. 2, 315-3m 3. Forrester P. I. and Hancock R. L. (1973) Can.J.Biochem. 51, 231-234. 0. D. and Kiselev L. L. (1968) Molekul.Biol. z>O-68. 4. Nelidova 5. Bartkowiak S. and Pawelkiewicz J. (1972) Biochim. Biophys.Acta 272, 137-140. 6. Remy P., Birmele’C. and Ebel J.-P. (1972) FEBS Lett. 2, 134-13’8T7. Hayashi H. (1973) J.Biochem. 74, 203-208. G., Remy P. and E&l J.-P. (1974) 8. Befort J. J., Befort N., Petrisszt Biochimie 56, 625-630. 9. Preiss J., Berg P., Ofengand E. J., Bergmann F. H. and Dieckmann M. (1959) Proc.Nat1.Acad.Sci.U.S. 45, 319-328. 10. Cramer F., von der Hoar F.and Schlimme E. (1968) FEBS Lett. 2, 136-139. 11. Zubay G. (1962) J.Mol.Biol. i, 347-356. 12. Zamecnik P. C., Stephenson M. L. and Scott J. F. (1960) Proc.Natl.Acad.Sci. U.S. 46, 811-822. 13. LamedR., Levin Y. and Wilchek M. (1973) Biochim.Biophys.Acta 304, 231-235. 139, 391398. 14. Wilkinson S. and Knowles J. R. (1974) Bi0chem.J. 15. Bonnet J. and Ebel J.-P. (1972) Eur.J.Biochem. 3135-344. 16. Hentzen D., Mandel P. and Garel J.-P. 0972) Bxchim.Biophys.Acta -’281 228-232.

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