Vol. 70, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMINO ACID ACCEPTOR ACTIVITY Arthur

Received

OF tRNA-C-C-C-A'

H. Kirschenbaum and Murray P. Deutscher Department of Biochemistry University of Connecticut Health Center Farmington, Connecticut 06032

February

25,1976

SUMMARY: Rabbit liver tRNA nucleotidyltransferase was used to synthesize modified tRNA molecules containing an additional CMP residue at the 3' terminus. In the first step tRNA-C-C was converted to tRNA-C-C-C by a minor activity of the purified enzyme. In the second step the lengthened molecules were converted to tRNA-C-C-C-A. AMP addition to tRNA-C-C-C occurred at about 50% the rate with tRNA-C-C. Aminoacylation studies indicated that tRNA-C-C-C-A was active for acceptance of at least 12 amino acids. INTRODUCTION: nucleotide

tRNA species

sequence,

acceptor Since

All

and transfer these

of these

logical

during activity

of value

for

tive

of tRNA molecules the

of modified

sugar

function

of this

nucleotides

or phosphate

Previous

to modification

work

from

tidyltransferase length

(3,4).

tRNA-C-A, acid

residues

this

could

acceptor

region

has shown

be used to synthesize

In an earlier

containing

one less activity

paper

it

terminal (5).

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

studying

that

terminal

In contrast,

258

sequence

of the regions

into

bio-

will

be

(1,2).

purified

that

tRNA by use of either

be to change

terminal

nucleotide,

modification protein-tRNA

to alteration

would

was shown

' This paper is number 15 in the series: Transfer RNA". The previous paper is

Copyright All rights

-C-C-A

the

common sequence.

leading of the

for

the aminoacyl-

examination 3'

tri-

references).

both

have been introduced

of this

laboratory

for

modified

1 for

of ribosomes,

In addition, with

identical

required

with

site

as a probe

synthesis.

is

(see

interact

transferase useful

the

which

molecules

enzyme tRNA nucleotidyltransferase

approach

amino

peptide

contain

3' terminus

presumably

prove

understanding

A variety

base,

nucleotides

should

examined

of these

and the peptidyl

residues

interactions

at the

functions

terminal

tRNA synthetases

of the

-C-C-A,

so far

liver

sequences

the

An alternaits

length.

tRNA nucleoof varying

one such tRNA molecule, was totally

we report

"Reactions at the reference 7.

devoid

here

that

of liver

3' Terminus

tRNA of

Vol. 70, No. 1, 1976

chains

with

variety

one additional

of amino

can accept

BIOCHEMICAL

AND BIOPHYSICAL

nucleotide

acids.

Thang

phenylalanine

residue,

--et al.

RESEARCH COMMUNICATIONS

tRNA-C-C-C-A,can

have,reported

and transfer

it

into

that

protein

accept

yeast

a

tRNAPhe-C-C-C-A

(6).

MATERIALS AND METHODS: Rabbit liver tRNA nucleotidyltransferase purified through step 6 was prepared as described previously (8). Rabbit liver tRNA and tRNA-C-C were prepared as reported earlier (9). Rat liver aminoacyltRNA synthetases were prepared by the procedure of Yang and Novelli (10) except that the DEAE cellulose and Sephadex G-100 chromatography steps were reversed. m-[32P] ATP and [14C] CTP were purchased from Schwarz/Mann. Radioactive amino acids were from New England Nuclear. Ribonuclease Tl was obtained from Worthington Biochemical. tRNA nucleotidyltransferase was assayed as described earlier (8). Details of individual experiments are described below and in the Legends. Conditions of the aminoacyl-tRNA synthetase assays are given in the Legend to Table I. tRNA-C-C-C-A was synthesized from rabbit liver tRNA-C-C by the sequential addition of CMP and AMP using purified tRNA nucleotidyltransferase. The structure of the modified tRNA was determined at each stage of the preparation by alkaline hydrolysis and measurement of nucleosides and nucleotides on Dowex-1 as reported previously (8 For this purpose the first product, tRNA-C-C-C was synthesized with [ I 4C] CTP. The reaction mixture (9 ml) ontained: 50 mM glycine-NaOH, pH 9.4; 5 mM MgC12; 15 mg tRNA-C-C; 0.5 mM [ F 4C] CTP (about 500 cpm per nmole) and 2.3 units of tRNA nucleotidyltransferase. The mixture was incubated for 3 hours at 37" and the rea ion stopped by After cooling, the tRNA-C-C-[ cz C]C product was puriheating at 60" for 5 min. fied on Sephadex G-25 containira silicic acid on top as described earlier (9). tRNA-C-C-A and tRNA-C-C-[ C]C-A were prepared by the addition of AMP to tRNA-C-C and tRNA-C-C-[14C]C. Conditions for AMP addition were essentially identical to those for CMP addition. In some experiments the time of incubation was shortened by the use of additional enzyme. RESULTS: showed

that

internal,

available

since

of the

75% of the

indicated

the

of 1.3

that

tRNA chains.

was solely

tRNA-C-C-[14C]C

incorporated

or an average

of termini

that

Analysis

product

[14C] residues

It

has not while

by alkaline

CMP residues per

chain.

CMP was incorporated

tRNA-C-C-C,

first

product

into

for

terminal

greater

all

further

the

and 25%

of the than

to synthesize

utilizing

was a substrate

were Quantitation

been possible still

hydrolysis

number

90% of the a preparation

available

tRNA chains,

CMP incorporation

by tRNA

nucleotidyltransferase. In order the rate the

second

to assess

step

and extent normal

at about corporation

of the

synthesis,

of this

reaction

substrate, 50% the into

the ability

tRNA-C-C

of tRNA nucleotidyltransferase the

incorporation

was compared (Fig.

1).

tRNA-C-C

(Fig.

rate

into

both

tRNAs reached

to that

1A).

However, limit

tRNA-C-C-C,

of AMP incorporation

AMP was incorporated

an identical

259

of AMP into

to catalyze the into

into

tRNA-C-C-C

the extent

of AMP in-

provided

that

sufficient

BIOCHEMICAL

Vol.70,No.1,1976

0

2

16

AND BIOPHYSICAL

8

loo

5

RESEARCH COMMUNICATIONS

10

15

20

MINUTES

Fig. 1. Comparison of rate and extent of AMP incorporation into tRNA-C-C and tk&C-C-C. (A) Reaction mixtures contained in 200 ~1: 50 mM glycineNaOH, pH 9.4; 5 mM MgC12; 0.5 mN [32P]ATP (about 1300 cpm/nmole); 1.6 ug of tRNA nucleotidyltransferase and either 80 pg tRNA-C-C or 100 vg tRNA-C-C-C. Samples were incubated for the indicated times at 37". (B) Reaction mixtures were identical to those in (A) except that they contained 110 pg tRNA-C-C with 8 ug enzyme or 80 pg tRNA-C-C-C with 32 ug of enzyme. Samples were incubated for the indicated times at 37".

enzyme was present

with

single

was incorporated

AMP residue For the large

scale

used as substrate, hydrolysis

[14C]CMP

residues

which

analysis

indicated

that

with

AMP.

AMP could

tRNA-C-C

extent

were

analyses

Further

evidence

voltage

electrophoresis

labeled

with

similar

pattern

[32P]

for

the

of the number

determined

that

greater

had already

accepted

AMP.

synthesis

of modified

upon electrophoresis,

the

260

first

labeled

were

cycle

by

termi-

than

of AMP in-

90% of the

tRNA was obtained

of tRNA-C-C-A Although

ATP was

how much more [22P]

indicated

AMP.

the

a

Such an

96% of the tRNA molecules we also

only

of terminal

residues.

after

terminal

unlabeled was determined

to penultimate

of RNase Tl digests in the

conditions

chains.

product

also

and tRNA-C-C-[14C]C

each of the

and measurement

In some experiments

These

these

of AMP incorporation

than

to the purified

Under

of tRNA-C-C-[14C]C-A,

converted

greater

1B).

into

of the product

be added

corporation.

(Fig.

preparation

and the

alkaline

nated

tRNA-C-C-C

by high

and tRNA-C-C-C-A

each of the

digests

3' oligonucleotides

gave a genera-

VoL70,No.

1, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I’

I Q :’ :: tRNA-C-C-C-A i\

1614 -

12p1 0

lo-

” 50

40

30

8

20

10

CENTIMETERS

FROM

0

10

8

ORIGIN

Fig. 2. Electrophoresis of ribonuclease Tl digests of tRNA-C-C-A and tRNA-C-C-C-A. tRNA-C-C-[32P]A and tRNA-C-C-C-[32P]A about 50 ug, were digested with 35 tig of Tl RNase at 37" for 30 min. Aliquots were spotted on Whatman 3 MM paper and electrophoresis run for 5.5 hours at 3000 volts in 0.02 M sodium citrate, pH 3.5. Strips of 2 cm were cut out and counted in toluene-based scintillation fluid.

ted

from

do not from

tRNA-C-C-C-A

identify

the

the modified

ses obtained the major

transferase

is also

to tRNA-C-C

(11).

before

consequence,

all

do show that which

Taken

of this

synthetic

scheme is

by as much as 30-40%

contamination This

but they

methods.

CMP residues.

be present.

action

product,

The electrophoretic

other

may be contaminated

a lo-15%

more slowly.

tRNA have been altered,

by the product

additional

migrated

However, with

heterogeneity

is

a poly(C) The presence the

chains

some unmodified

is

with

amino

due to the

these

with data

tRNA-C-C-C-A. higher

fact

liver

which

can add multiple

of this

activity

necessitates

chains

still

261

more than remain.

that

containing

that,

polymerase

have added

analy-

The preparation

acceptor, that

the

indicate

homologues

indicates acid

alone

3' oligonucleotides

consistent

together,

our analysis

the normal

the

data

at most,

tRNA-C-C-A,

could

tRNA nucleotidylCMP residues stopping

one CMP residue, Nevertheless,

only

the

but as a this

prepara-

re-

Vol. 70, No. 1,1976

tion

is

with

additional

tively

satisfactory

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

determining

the

CMP residues

since

to that

of amino

of tRNA-C-C-A

The tRNA-C-C-A to tRNA-C-C served

with

for

acceptor

activity

by unmodified

for

pared

of tRNAs

tRNA is

rela-

histidine,

tRNA,

at low

levels

Another

group

poration

is

taining

close

account

in Table

acids

to the three for

of normal

tRNA.

acids

tested

greater

than

would

be expected

C-C-A.

For this

as substrates

of liver at the

it

presented

a number

into

This

acceptors

levels

(20-40%),

in-

suggest

that

262

This

partial

tRNAs that

can tolerate contrasts

still

the

paper

tRNA.

but

of tRNA-

whether

results

they

preparation

in this

modified

since

con-

The remainder

incorporation.

synthetases

of incor-

the tRNA chains

maximal

These

tRNA.

and isoleucine)

for

acids.

namely

tRNA-C-C-C-A

extent

if

isoaccepting

com-

by normal

present.

certain

of the

acids,

conditions

of amino

aminoacyl-tRNA 3' end of the

not yet

amino

of the

tRNA-C-C-C-A.

tRNA-C-C-C-A

proline

not

to lower

contamination

a fraction

or to inadequate

for

is

were

tRNA chains

incorporated from

group

due to only

DISCUSSION : The data as an acceptor

were

total

used for

into

be expected

CMP residues

of the

determined

first

contamination

of 40-60%

of tRNA-

were

incorporated

valine,

terminal

amount

which

concentration

were

leucine,

amino

is

acids

This

and enzyme

of the

might

of AMP

oxidation

the

also

(alanine,

of the

corporation

acid were

to the

addition

periodate

Several

acid

synthetases.

of tRNA-C-C-C-A.

Since

amino

I.

maximum that

30-40%

latter

conditions

be attributed

to levels

more than

probably

these

and aspartic

could

of amino

incorporated

by the

of time

of various

is shown

which

(5).

of each amino

and then

acid

acids

the conditions

incorporation

glutamic

was prepared

of some amino

tRNA was compared

aminoacyl-tRNA

to the preparation

incorporation

to tRNA-C-C-A

liver

any damage to the tRNA during

maximal

The relative

of rat

experiments

was limited,

unmodified

by the lengthened

a mixture

identical

acceptance

available

required

bility

acid

contamination

acceptance

using

in a manner

affect

C-C-C-A

acid

used in these

to correct

could

variety

amino

low. The extent

were

BIOCHEMICAL

are active

tRNA-C-C-C-A indicate the

that

increased

with

the

can act a flexi-

loss

of

BIOCHEMICAL

Vol. 70, No. 1, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I Extent Amino ----

of Aminoacylation

of tRNA-C-C-C-A

Acid

Compared Relative

to tRNA-C-C-A incorporation

aspartic

acid

0.12

glutamic

acid

0.13

histidine

0.13

arginine

0.21

lysine

0.24

threonine

0.26

tyrosine

0.28

serine

0.30

methionine

0.32

glycine

0.37

proline

0.45

alanine

0.46

leucine

0.48

isoleucine

0.49

valine

0.54

Aminoacylation was measured in reaction mixtures of 100 ~1 containing: 0.25 M Tris-Cl, H 7.0, 5 mM ATP; 5 mM MgCl ; 10 ug bovine serum albumin; 0.2 mM EDTA; 0.25 mM [ H or [14C] amino acid (5- f 0 cpm/pmole); 50-100 ug tRNA-C-C-A or tRNA-C-C-[1 9 C]C-A and 40 ~1 of mixed rat liver aminoacyl-tRilA synthetases. Reaction mixtures were incubated 10 to 20 min at 37" and stopped by the addition of 10% TCA containing 0.02 M sodium pyrophosphate. The precipitate was collected on GF/C filters, washed, dried and counted. Zero time blanks were subtracted from each assay. Incorporation is expressed relative to that into tRlU-C-C-A which is set at 1.00. Incorporation into tRNA-C-C-A varied from 0.2 to 2.5 nmoles/mg depending on the amino acid.

activity

of tRNA-C-A

reported

here

flexibility suggest

with

purified

at the that

for

chains

(5).

It

would

tRNA species

3' end of tRNA might

several

amino

acids

be of interest to determine

lead

not

263

all

to repeat whether

to mischarging. of the

isoaccepting

the

the studies increased

Our results species

of

Vol.70,No.1,1976

tRNA-C-C-C-A of both

BIOCHEMICAL

are

an extra

active. CMP residue

the molecule

could

lead

of activity.

to loss

terminal

sequence

ACKNOWLEDGMENT: tance.

This

work

tutes

of Health.

This

render

with

raises at the

the

possibility

3' terminus

a tRNA inactive, This

other

We thank

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

parts

Sherrie

was supported

the

and a change

whereas

may indicate

that

either

interaction

combined

effect

in another

change

alone

of residues

part

does not in the

of the tRNA molecule. Perry

by grant

for

her excellent

GM-16317

from the

technical

assis-

National

Insti-

REFERENCES

:: 3. l: 6. 2 1:: 11.

Deutscher, M.P. (1973) Prog. Nucleic Acid Res. l3-, 51-92. Sprinzl, M., Scheit, K., Sternbach, H., von der Haar, F. and Cramer, F. (1973) Biochem. Biophys. Res. Commun. g, 881-887. Deutscher, M.P. (1972) J. Biol. Chem. 247, 469-480. Deutscher, M.P. (1973) J. Biol. Chem. E, 3116-3121. Tal, J., Deutscher, M.P. and Littauer, U.Z. (1972) Eur. J. Biochem. 28, 478-491. Thang, M.N., Dondon, L., Thang, D.C. and Rether, B. (1972) FEBS Letter 26, 145-150. Morse, J.W. and Deutscher, M.P. (1975) J. Mol. Biol. 95, 141-144. Deutscher, M.P. (1972) J. Biol. Chem. 247, 450-458. Deutscher, M.P. (1972) J. Biol. Chem. m, 459-468. Yang, W.K. and Novelli, G.D. (1968) Proc. Natl. Acad. Sci. U.S.A. 59, 208-215. Deutscher, M.P. (1973) J. Biol. Chem. 248, 3108-3115.

264

of

3'