Inactivation of the Tu-GTP recognition site in aminoacyl-tRNA by chemical modification of the tRNA

Vol.41,No.

BIOCHEMICAL

1,197O

INACIIVATION

OF THE Tu-GTP

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RECOGNITION SITE IN AMINOACYL-tRNA

MODIFICATION Roche

Received

OF THE tRNA

Chong-Maw Chen and James Ofengand of Molecular Biology, Nutley, New Jersey

Institute

August

BY CHEMICAL

07110

31, 1970

SUMMARY: Modification of tRNA Phe of Brewer's yeast by the technique of periodate oxidation, borohydride reduction to the diol, and enzymatic charging with phenylalanine yielded a Phe-tRNAoXmred whose 2',3' carboncarbon bond at the 3'-terminal ribose was absent. This modified Phe-tRNA was unable to form a ternary complex with Tu-GTP when assayed by the Millipore filter technique and was unable to bind to ribosomes at either the T factor dependent or independent sites. This represents the first example of a modification at a specific locus in the tRNA molecule which affects the Tu-GTP recognition site. INTRODUCTION: synthesis

The role

of the T factors,

has become

clear

in recent

(2-8),

and is

summarized

laboratories

by the Tu-GTP-AA-tRNA1 polypeptide

synthesis

mechanism

operates

synthetic

machinery.

binding

complex

to exclude

normal

with

Tu and GTP (g-13),

group

peptide

has been

show that

certain

complex

linked

tRNA.

although

to the it

carries

selection

emphasized unwanted

by studies

tRNAs and AA-tRNAs

site

because

they

and the

same is

by nitrous

acid

First, a normal

to the

acid,

1 Abbreviations

are

played

AA-tRNA

for

from

the protein

are prevented

cannot

for

role

show how this

and hence

(13). also

from

form

AA-tRNAs

treatment

Met-tRNA fMet amino

AA-tRNAs

true

of tRNA structure

in addition

which

the

whose

from interfering initial amino

complex acid

Two additional required

for

presence

of an unmodified

does not

interact

and second,

protein

of several

The central of a proper

ribosomal

formation

in bacterial

the efforts

1.

requiring

aspects

by the Tu-GTP

in Fig.

or N-acyl

bond

removed

through

Deacylated

at the T factor

with

years

in the

has been

Tu and Ts (1)

the

with denatured

studies

recognition amino Tu-GTP

acid (14)

form of

used are: AA-tRNA, aminoacyl-tRNA; Met-tRNA fMet , $,t";;;y'tRNA species which can be formylated; Leu-tRNA, leucyl-tRNA; tRNA Phe-tRNA, tRNA sggc:pi accepting phenylalanine and phenylalanyl-tRNA, respectively; Phe-tRNA , phenylalanyl-tRNA chemically modified as described in the text.

190

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 41, No. 1,197O

AA-tRNA

+ Tu-GTP

TV-GTP-AA-tRNA RIEOSOMES m-RNA

GTP TS

Tu-GDP + Pi

\

x AA-tRNA-RIBOSOME-mRNA + POLYPEPTIDE

Fig.

1. The Tu-GTP-AA-tRNA Weissbach --et al. (7).

Leu-tRNA

of yeast

interacts

an unmodified

amino

As part

of our

in this

yeast

is

diol

studies

treated

first

moiety

is

phenylalanine

and a crude et al. procedure

cannot Phe-tRNA

EXPERIMENTAL: pmoles

with

yeast

(17). for

with

react

Phe-tRNA

with

the.Tu-GTP

phenylalanine

oxidation

of both

by addition

from

too possesses

of a 1.5-fold

amino

tRNA, we report

acid

borohydride,

the this

- Brewer's

yeast

was obtained

as described

acceptance,

and was isolated

excess

191

adenylate

from Boehringer,

up the standard

temperature

in

tRNA Phe (1200

by scaling

2-200

resulting

Phe-tRNA

of the terminal

was prepared

pH 5.4;

esterified

When tRNAPhe of

However,

G-25 gel

tRNA Phe and Phe-tRNAPhe

room

in

complex.

and Sephadex

0.1 M NaOAc buffer, 30 min at

with

ribose

unit)

measuring

Periodate

minated

it

complex.

(16).

of the

was synthesized

site

the Tu-GTP

bond

Phe-tRNA

for

adapted

15) although

a normal

phenylalanine

synthetase

precipitation,

mM NaI04

(13,

and then

acceptanceJA260

ethanol

l-4

synthesis

recognition

and Phe-tRNA ox-red

treatment,

containing

with

periodate

carbon-carbon

absent

poorly

case in which

interact

be charged

the 2',3'

very

on the Tu-GTP

a third

tRNA cannot

can still

which

only

in protein

acid.

communication

to a modified

cycle

filtration

was performed

A26o units in the dark.

of glucose

by Lindahl assay by phenol (18). in a solution

per ml of tRNA, Oxidation

to periodate.

and

was terThe oxi-

VoL41,No.

dized

1,197O

tRNA was isolated

in water.

phosphate acetic

buffer,

dark.

solution

Other

for

both

above,

conditions. both

This

1).

materials

procedure

- Uniformly

which

2.

['H]GTP

Research.

Poly

and Ts were

generously

Ribosomes

bath. dure

U was obtained

described

enzymatic times kindly

by Ofengand

by Mr.

Phenylalanine as previously

The filters

(19).

(18)

413 100,000

Inc.

phase

E. coli that

were

BioTu

and H. Weiss-

413 by the proce-

ribosomes

used

additionally

x g supernatant

Schwarz

95% pure

J. Hachmann,

except

binding

from

mcfmmole

for

washed protein

3

was

2. Hussain.

acceptance (18) were were

log

the same

of 36.8

was purchased

D. Miller,

to

was obtained

activity

Laboratories,

late

and Henes

E. -coli

described

20 PM, and reactions

from

14 C-Phe-tRNA

0.5 M NH4Cl.

supplied

by Drs.

prepared

or nonenzymatic

with

from Miles

supplied

were

Ci/mmole)

was

was carried

in Fig.

in all

(1.16

and reisolation

C-L-phenylalanine

it

exposed

summarized

and used at the same specific

experiments.

had been

to almost

from Amersham/Searle

alka-

the Phe-tRNA,

charged

14

in

precipitation,

being

labeled

of oxidized

was sufficiently from

Recharging

is

to one volume

reaction

preparations

with

110 min at room temperature

the tRNA and Phe-tRNA

and reducing

of NaBH4 in 0.2 M

once by ethanol

reduction

essentially

at room temperature

was added

and washed the

solution

to pH 8.1

and incubated

Since

to recharge

(Table

A 5 mg/ml

and dissoived

was performed

70% or more of the phenylalanine

as described

extent

of this

precipitation,

to the diol

was adjusted

in water.

to hydrolyze

once by ethanol

(16).

The tRNA was isolated

the oxidizing out

pH 7.8,

A260 units/ml)

and dissolved

necessary

et al.

One volume

tRNA (25-120

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the dialdehyde

by Cramer

acid.

line

and washed

Reduction

as described

the

BIOCHEMICAL

was assayed except

stopped dissolved

the

in the

absence

of C

14 C-phenylalanine

by TCA precipitation

12

amino

acids

concentration on Millipore

in naphthalene-dioxane

solution

was filters

(20)

and

counted. The formation retention

of Tu-GTP and Phe-tRNA-Tu-GTP 3 of the Tu-[ H]GTP complex on Millipore

192

complexes filters

was assayed

by

and the reduction

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 41, No. 1,197O

in this

retained

ternary

complex

first

radioactivity through

converted

containing

the

to Tu-GTP

to 0';

The reaction

Phe-tRNA

a reaction

80 mM KCl;

binding,

Incubations

for

of a wash buffer After

MgC12. ter,

the

was washed

incubated

three

(20)

times

mixture

with

5 min at 0'. (7).

was measured pH 7.4;

1 mM dithiothreitol; kinase;

60 units

Tu

of 0.5 M NH4Cl 14

of

from the

C-Phe-tRNA. reaction

by the addition

pH 7.4;

then

50 mM Tris-Cl,

amounts

and terminated

50 mM Tris-Cl,

the diluted

in naphthalene-dioxane

ml,

omitted

(7),

conditions.

U; 3.3 A26o units

413 and various

15 min at 30°,

passing

these

to ribosomes

ug pyruvate

Tu, Ts and GTP were

containing

filter

coli

buffer

previously

under

of 0.15

poly

for

as described occurs

in a volume

kinase

incubated

of Phe-tRNA

1.1 A26o units

from g.

For nonenzymatic were

binding

0.7 II% GTP; 0.04

Ts (8);

70s ribosomes

processed

Tu-GDP was

in assay

15 mM or 8 mM MgC12 as indicated;

2 mM phosphoenolpyruvate; 1800 units

(7).

10 min

and the mixture

or Phe-tRNA ox-red

containing

80 mM NH4Cl;

previously

at 37O for

and samples

and nonenzymatic mixture

of the Phe-tRNA-Tu-GTP

and 20 pg of pyruvate

was added,

of Phe-tRNA

Enzymatic

washed

as described

by incubation

was stopped

No hydrolysis

(8);

filter

7 mM phosphoenolpyruvate

chilled

in

due to the passage

mixture.

of 1.0 ml

160 mM NH4C1, and 12 mM through

2 ml of cold

a Millipore

buffer,

fil-

dissolved

and counted. TABLE 1

PHENYLALANINE ACCEPTANCE ACTIVITY

AFTER OXIDATION Specific

Prep. (1) Original Periodate-treated

(3)

NaBH4 reduction Preparation described

Activity

(pmoles/A260)

RNAPhe

tRNA Preparation

(2)

Phe AND REDUCTION OF tRNA

of

(2)

of modified in Experimental.

A

Phe-mAPhe Prep.

B

Before Recharging

After Recharging

1242

1159

1165

1330

<7

<6

1010

1040

916

908

317

1135

tRNA and assay

193

of acceptor

activity

was as

Vol. 41, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS: Preparation of Phe-tRNAox-red - Table 1 summarizes the acceptance activity

of the tRNA at each stage of modification.

the only relevant

modifications

were those occurring

sine group, a pre-charged control ment since oxidation

at the 3'-terminal

adeno-

Phe-tRNA was also carried through the treat-

at the 3'-end of this molecule is prevented by the pres-

ence of the amino acid.

Line (2) of the table shows that periodate oxidation

was complete since no acceptance activity and line

In order to confirm that

was left

in the tRNAPhe preparation,

(3) shows that subsequent reduction almost completely restored the

acceptance activity

(16).

bound before and after

The slight

in the amount of phenylalanine Phe the recharging of Phe-tRNA in the activity assay

probably is due to somedeacylation since after

periodate treatment,

of the oxidation-reduction

during the preparation

this difference

reaction

because (a) the above functional

difference

was abolished.

is assumedto be as indicated

effects

,,.,YOY 742

H27

HO

OH WITH

CHARGE

PHE

1

tRNAYoY 7J.42

The product in Pig. 2

are in agreement with those previously

tRNA

H27

0

0

I Phe

Fig. 2.

of this material

Preparation of Phe-tRNAox-red .

194

Vol.41,No.

BIOCHEMICAL

1,197O

reported

by Cramer

et al.

and (b)

the half-life

for

Tris

buffer,

reaction

pH 8.6,

obeyed

(21,

expected

other

sites

the Phe-tR.NA functional,

in the control they

Reaction complex

These

with

of complex

Tu-GTP formed

for

tRNA.

is

kinetics.

However,

if

diol

in the

ox-red

vs.

stoichiometric

PHE-tRNA

a cis-diol

3.

occurred

material

discussed

In this

with

and the is ester

reactions

must have

of Phe-tRNAoXSred

in Fig.

in 0.1 M

at the aminoacyl-end

this

changes

at 37'

in reactivity

modification they

reaction,

Phe-tRNA

decrease ester

and since

- The failure

illustrated

exclude

present,

also,

be involved

was almost

This

of this

normal

has occurred

do not

pMOLES

Fig.

to 16 min for

a modification

preparation

Tu-GTP

of Phe-tRNA

an unoriented

observations

the product

hydrolysis

order

that

cannot

with

chemical

first

22) and confirms

of the tRNA.

wh o characterized

was 52 min compared

strict

in the direction

(16)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

remained here.

to form experiment,

the amount

at

a ternary the amount

of AA-tRNA

added,

ADDED

3. Ability of Phe-tRNA and Phe-tRNAoXmred to react with Tu-GTP complex. The formation of Phe-tRNA-Tu-GTP ternary complex was assayed by the Millipore filter technique as described in Experimental. In this composite of three experiments, 100% GTP retained on the filter ranged from 20.6 to 34.0 pmoles. Untreated Phe-tRNA,O; Phe-tRNA exposed to the oxidation and reduction conditions, a; and Phe-tRNAoXmred (two different preparations),Dand A.

195

Vol. 41,No.

1,197O

BIOCHEMICAL

as shown

by the open

Phe-tl?NA

carried

however,

is

if

this

0, 7.2,

15,

normal

likely since with addition have

unable

and 22.5

that that

it

mixtures

was released

no inhibitory

treatment,

of 22.5 the would

then

that as the

released

r3H]GTP

have been

were

but

from

readily

8 mM

were

added

with

formed

which

Phe-tBNA

Tu-GTP.

7 to 4.6

pmoles

To see

preparation,

pmoles

of

of Tu-GTP.

was present Moreover,

it

was not bound

of normal or less.

or not, is

un-

filterable,

to Tu-GTP

made a non-filterable to 9.8 pmoles

aud

Phe-tENAoXVred,

to 9.8 pmoles

15.2

present.

the modified

control

with

the Phe-tENAoX-red

of Phe-tEEA ox-red

pmoles

complex

complex

if

Phe-tENA

respectively.

reacted

whether

-Tu-GTP

untreated

in the Pbe-tENA ox-red

substances

a Phe-tENA ox-red

the same affinity

of reduction

chemical

of Phe-tENA ox-red

pmoles

can be calculated

reduced

representing

to make a filtrable

and these

of [3H]GTP

indicating

the

circles,

was due to some inhibitor

Phe-tENA

7 pmoles

through

clearly

failure

and filled

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

complex,

the

tEEA should This

amount

detected.

Mg++

20 F-

pMOLES

Fig.

PHE-tRNA

ADDED

of Phe-tENA and Phe-tENAoX-red 4. The binding to ribosomes with and without Tu, Ts and GTP. Each of the 0.15-ml reaction mixtures contained the components indicated under Experimental. (A) at 8 mM Mg*, (B) at 15 mMMg*. Oand 0, Phe-tENA e osed to the oxidation and reduction conditions;aand Ar Phe-tf(NAox-re 3 . Open symbols, with Tu, Ts and GTP; filled symbols, without Tu, Ts and GTP.

196

Vol.41,No.

BIOCHEMICAL

1, 1970

Ability

to bind

to ribosomes

AA-tRNA-Tu-GTP

complex

dered

that

the

possible transfer

especially might

different

from

4, this

binding

abolished,

but

binding

to the peptidyl-tRNA

control

Phe-tRNA

carried

through

In other

words,

selecting

the Tu-GTP

that

to block

interaction

these

by its

two classes The Tu-GTP

the minimum correct

(a)

It

tertiary

adenosine should

in these

only

the

that

had been

utilized

the

by the Tu-

by aminoacyl-

AA-tRNAs

these

on

not shown).

are recognized

those

active

state.

dependent

(data

must

not be

same differences

of a mixture out

that

of similar

are

molecules.

common features

of AA-tRNAs,

among individual

tRNA mole-

carbon-carbon complex

emphasizes

but

bond did

not

of tRNA

Phe

effect

the difference

between

sites.

site

amino

Note

system

that

case,

the Tu-GTP

to represent

in the precharged

different

of the 2’,3’

dependent

in Experimental

differences

synthetase

of recognition

a free

the

with

recognition

overall

terminal

out

seeks

cleavage

cognate

latter

However,

thought

from case,

recognition

binding,

to function

tRNAs out

complex

search

Our finding

recognition

in the

allow U complex,

was T factor

tRNA molecule

cognate

the synthetases

was able

used

while

Tu-GTP

synthesizing

former

was consi-

might

Not only

treatment

different

in the

it

to the ribosomes.

described

was unable

of the

since

for

(4), was also blocked.

site

be distinctly

against, for

case.

independent

ox-red

Phe-tRNA

must

essential

cules.

T factor

The features

discriminated

the

no stable

Phe-tRNA-ribosome-poly

binding

in a polyphenylalanine

tRNA synthetases,

while

was not

for

the oxidation-reduction

Phe-tRNA,

GTP complex

that

assay,

of such a complex

a stable

was the preparation

When tested

DISGUSSION:

formation into

- Although

by the filter

of the tRNA responsible

in Fig.

added

be detected

ox-red

the area

be quite

and make polypeptide

the transient

of Phe-tRNA since

as shown

could

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

group

in AA-tRNA

can now be said

on the esterified

structure,

and (c)

amino

an intact

to require acid,

ribose

ring

(b)

at a

of the 3’-

group.

be emphasized experiments

that had been

all

of the

exposed

197

fully

to the

active

control

same chemical

Phe-tRNA treatment

as

VoL41,No.

1,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the test tRNA except that the 3'-terminus of phenylalanine. to the activity modifications

Therefore,

was blocked by enzymatic charging

the only chemical change that can be relevant

differences

between the two preparations must be related to

at the 3'-end.

While we have so far presented only indirect

evidence that such a change occurred, preliminary zation of the phenylalanyl-adenosine

derivatives

by pancreatic RNasedoes confirm that a structural

chromatographic characteriliberated

from the Phe-tRNAs

change took place.

Experiments are now underway to replace the modified terminal adenylate group by a normal one in order to demonstrate more directly chemical treatment affected modifying the 3'-terminus

the Tu-GTP complex forming ability

that this only by

of the tRNA. REFERENCES

Lucas-Lenard, J., and Lipmann, F., Proc. Nat. Acad. a., E.g., 55, --1562 (1966). 33, 264 (1969). 2. Lengyel, P., and S&l, D., Bacterial. g., Lipmann, F., -9 Science -9 164 1024 (1969). 2: Ravel, J. M., Shorey, R. L., Garner, C. W., Dawkins, R. C., and Shive, W., Cold Spring Harbor Sump. Quant. Biol., 34, 321 (1969). D., Rossman,M., and Ertel, R., m 5. Weissbach, H., Brot, N., Miller, Spring Harbor Svme. Quant. Biol., ,,34 419 (1969). 6. Skoultchi, A., Ono, Y., Waterson, J., and Lengyel, P., Cold Spring Harbor Sm. Quant. Biol., 34, 437 (1969). D. L., and Hachmann, J., PP Arch. Biochem. Biophvs., 7. Weissbach, H., Miller, 137, 262 (1970). 8. Miller, D. L., and Weissbach, H., Biochem. Biophys. Res. Commun.,38, 1016 (1970). 9. Ravel, J. M., Shorey, R. L., and Shive, W., Biochem. Biophvs. Res -* Coxmaun ., 29, 68 (1967). E. Acad. E., E.S., 59-, 179 (1968). 10. Gordon, J., s. H. M., and Lengyel, P., Proc. Nat. &&. 11. Skoultchi, A., Ono, Y.,sn, -m &&., g.g., 60, 675 (1968). Ertel, R., Redfield, B., Brot, N., and Weissbach, H., -Arch. Biochem. 12. Biophvs., 128, 331 (1968). 13. Jeres, C., Sandoval, A., Allende, J., Henes, C., and Ofengand,J., Biochemistry, g, 3006 (1969). 14. Ono, Y., Skoultchi, A., Klein, A., and Lengyel, P., Nature, 220, 1304 (1968). 15. Chen, C.-M., and Ofengand, unpublished experiments. 2 136 (1968). 16. Cramer, F., v.d. Haar, F., and Schlimme,E.,FEBSLetters --* -9 Chem 242 3129 (1967). 17. Lindahl, T., Adams, A., and Fresco,J.R.,J..BioL.-09-s 18. Ofengand, J., and Henes, C., 2. Biol. a., 244, 6241 (1969). Methods in 19. Scott, J. F., in S. P. Colowick andN. 0. Kaplan (eds.) -Enzymology, Vol. KIIB, Academic Press, NewYork, 1968, p. 173. 20. Bray, G. A., Anal. Biochem., 1, 279 (1960). 21. Bruice, T. C.7 and Fife, T. H., J. 9. &. &., 84, 1973 (1962). 22. Zachau, H. G., Feldmann, H., Frank, W., Karau, W., and Sonnenbichler, J., in P. M. Bhargava (ea.) Nucleic Acids Structure, Biosynthesis and p-0 Function, New Delhi, India, Council of Scientific and IndustriaEesearch, (19651, pp. 238-245. 1.

198