Inhibition of the peptidyl transferase A-site function by 2′-O-aminoacyloligonucleotides

Vol.56,No.3,

1974

BIOCHEMICAL

INHIBITION

AND BIOPHYSICAL

OF THE PEPTIDYL

RESEARCH COMMUNICATIONS

TRANSFERASE A-SITE

FUNCTION

BY 2’-O-AMINOACYLOLIGONUCLEOTIDES David Michigan

Received

December

Ringer

Cancer

and Stanislav

Foundation,

Chla/dek*

Detroit,

Michigan

48201

11,1973 SUMMARY

New "non-isomerizable" analogs of the 3'-terminus of AA-tRNA, C-A(2'Phe)H, C-A(P'Phe)Me, C-A(2'H)Phe and C-A(2'Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3'-O-AA-derivatives were active acceptors of Ac-Phe in while the PI-O-AA-derivatives were completely the peptidyl transferase reaction, inactive. Both 2'- and 3'-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3'-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3'-terminus of 2'-OAA-tRNA. INTRODUCTION Since

the discovery

in protein

biosynthesis,

terminal

adenosine

Because the

of the

cis-diol

the

Moreover,

tRNA that

is

unit

the

employed

* To whom reprint 110 E. Warren Avenue,

direct

exact

involvement

location

has been the rapid

terminal

exact it

the

the

extremely

of

to determine tRNA.

of

2'

subject

z 3'

of

now appears

that

throughout

the

the

of

unit

the

of

(2,3),

aminoacyl it

is

various

requests should Detroit, Michigan

amfnoacyl

considerable

migration

adenosine

position

of

of 2'(3')-U-aminoacyl-tRNA

not

the it

is

residue a single

stages

be sent: 48201.

on the

interest

(1).

aminoacyl

group

almost

impossible

in native isomer

of protein Michigan

residue

of

of

aminoacylaminoacyl-

biosynthesis.

Cancer

Foundation,

Abbreviations: C-APhe, cytidyly1(3'-5')-2'(3')-O-L-phenylalanyladenosine (I); C-A(2'Phe)H, cytidyly1(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa); C-A(2'Phe)Me, cytidyly1(3'-5')-3'-O-methyl-2'-u-~-phenylalanyladenosine (IIb); C-A(2'H)Phe, cytidyly1(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa); C-A(2'Me)Phe, cytidyly1(3'-5')-2'-&methyl-3'-O-L-phenylalanyladenosine (IIIb); tRNAox-red, tRNA w ich has been oxidized with periodate, reduced with borohydride (6); A#,;'" !4 , L-phenylalanyl derivative of oxidizedreduced adenosine, either "2"' or "3"' -isomer (for correct nomenclature of the latter, see ref. 5). 760

Vol.

56, No.

On the

3, 1974

basis

BIOCHEMICAL

of the

amtnoacyl-tRNA inhibiting

structure

and its

AND

and activity

2'-analog

polypeptide

BIOPHYSICAL

(the

synthesis),

of puromycin

latter

it

RESEARCH

COMMUNICATIONS

as a 3'-analog

was found

of

to be inactive

has been suggested

that

in

3'-O-aminoacyl-

C

HOCHz

0

1cs; 0 7 HO-P=0

0

OH

I HO-P=0

OH

OH

HO-P=0

I A

&HZ

6Cl-l~

A

0 w

ii

bPhe

PheO

R

v Phe.H

I

n

o;R=H

IItO,R=H

b,R=OCHa

tRNA is

the

active

we have

recently

analog

of

ferase

reaction

reported

is

3'-isomer

PI-isomer

is

formed

by the

(4).

acceptor

must occur

peptidyl

inactive (7)

have

synthetase after

a "non-isomerizable" in the

virtually

and Cramer

AA-tRNA

More specifically,

of AFi,red,

an active

and Sprinzl

transacylation

(5).

shown

reaction,

enzymic

transStudies

that

thus

2'-Oindicating

aminoacylation

and before

bond formation. As part

various

whether

several the

(ITb), substrates

of our stages

or not

ribosomal

at

biosynthesis

the is

its

and Chen (6)

2' + 3'

the

that

and that

aminoacyl-tRNA

peptide

in protein

3'-U-aminoacyl-tRNA,

by Ofengand

that

isomer

bj R=OCHa

study

of

of protein

a 2'

+ 3'

or 3'-hydroxyl.

C-A(2'H)Phe for

(IIIa) peptidyl

this

in which compounds,

and C-A(2'Me)Phe transferase

3'-terminus

is a required

to study

These

of the

we have been

transacylation

aminoacyloligonucleotides 2'-

involvement

biosynthesis,

In order

mechanism.

the

the

and as inhibitors

761

residue

(IIa),

C-A(2'Phe)H

(IIIb),

in the

we have

aminoacyl

were

tested of

tRNA in

interested

step

question,

of

peptidyl

to learn overall

synthesized is

"fixed"

C-A(2'Phe)Me as acceptor transferase

Vol.

56,

No.

3, 1974

catalyzed

BIOCHEMICAL

Ac-Phe

3'-0-aminoacyl

to

center,

of

both

peptidyl

aminoacyl-tRNA

BlOPHYSlCAi

puromycin.

(IIIa,

derivatives

transferase bitors

transfer

AND

2'-

Our results

IIIb)

can act

show that

that

by the

peptidyl

the

although at

derivatives

indicating

can be recognized

COMMUNICATIONS

as acceptors

and 3'-U-aminoacyl

transferase,

RESEARCH

the

peptidyl

are potent

3'-terminus

of

transferase

only

inhi-

2'-O-

A-site.

MATERIALS AND METHODS Three

times

Biochemicals)

NHqCl-washed

and Ac-(3H)Phe-tRNA,

alanine/mg

tRNA, were

The chemical phosphates

the

in this

assay

study

2'-

will

0.44

described

cells

nmoles

(General

phenyl-

(5).

and 3'-O-aminoacyldinucleoside elsewhere.

was measured

are given

MRE-600

activity

be described

activity

conditions

E. coZi

as previously of the

transferase

from

specific

prepared

synthesis

used

Peptidyl of

ribosomes

in the

as before

figure

(5).

Details

legends.

RESULTS AND DISCUSSION As can be seen

(IIIa)

C-A(2'H)Phe

from

Fig.

1, only

peptidyl

transferase

reaction

system.

No activity

has been observed

range

adenosine

derivatives

acceptor

of

1 X 10m5 M less

than

report

by the of

peptidyl

transferase

proved

that

prepared 3'-A$red

reaction, by chemical indeed

These

whereas

active

762

over

a wide

with

the

analogous

The decreased

to C-APhe

(I)

may be attributed results

a mixture

synthesis

P'-O-amino-

(IIb)

same trend

relative

are

that

(8) (at to

consistent

2'-A$red,

was active. acceptor

the

2'-

with

in the

and 3'-isomers

Our recent (5).

steric

prepared

was inactive of

in the S ribosome

corresponding

and digestion,

the

of Ac-Phe

elsewhere).

(9) who showed

aminoacylation

is

the

the

of C-APhe)

group.

acceptors

and C-A(2'Phe)Me

(IIIb)

that

and Ofengand

by enzymic

ox-red Of APhe

50% of

with

observed

C-A(2'Me)Phe

derivatives

Ac-Phe-tRNA*poly(U)*70

to be published

2'-O-methyl

Hussain

tRNAoXmred

We have

are

the

(IIa)

(results

activity

hindrance

using

C-A(2'Phe)H

of concentration.

3'-O-aminoacyl

(IIIb)

and C-A(2'Me)Phe

acyloligonucleotides

the

report

We therefore

the from

Vol.56,No.3,

1974

BIOCHEMICAL

100

AND BIOPHYSICAL

r

ACCEPTOR

Figure

1

conclude of

the

that

peptidyl

transferase

although

this

fact

of AA-tRNA

is

required

ribosomal Binding

peptidyl

CONC.

(M)

2’- and 3'-O-L-Phenylalanyldinucleoside phosphate dependent release of the Ac-(3H)Phe residue from Ac-(3H)Phe-tRNA in the peptidyl transferase reaction. Each reaction mixture contained in 0.10 ml: 50 mM Tris-HCl (pH 7.4), 100 mM NH4C1, 10 mM MgC12, 3.7 A2 0 units of ribosomes, 10 pg poly(U), and 0.12 A260 units of AC-(3H P Phe-tRNA (2000 cpm) . The reaction was initiated by addition of the acceptor compounds at the concentrations indicated. Following incubation at 37' for 30 min, the reaction was terminated by the addition of 2.0 ml of 2.5% CC13COOH at 4'. After 15 min at 4", the entire reaction mixture was filtered through a HAWP-Millipore membrane which was then washed with three 2.0 ml portions of cold 2.5% CC13COOH. After the membranes were dried, the radioactivity was determined in a 4.5 g PPO/lOO mg dimethyl-POPOP/l liter toluene scintillation mixture. The amount of Ac-(3H)Phe residue transferred from Ac-(3H)PhetRNA to the acceptor was determined as the difference between radioactivity retained on the filter after incubation without acceptor and that retained after incubation with an acceptor. It was expressed as the percentage of the radioactivity of Ac(3H)Phe-tRNA added to the experimental mixture. Symbols: 0, C-APhe (I); A, C-A(2'Phe)H IIa); 0, C-A(2'Phe)Me (IIb); A, C-A(2'H)Phe (IIIa); m, 2'Me)Phe (IIIb).

AA-tRNA,

3'-isomer

RESEARCH COMMUNICATIONS

possesses per

se does for

all

specificity

for

the

3'-isomer

not

necessarily

mean that

steps

of protein

biosynthesis

the at

stage.l of

transferase

the

acceptor A-site

terminu-s should

of AA-tRNA

precede

the

peptide

1 Recently, Fraser and Rich (10) speculated newly-formed peptidyl-tRNA (which is the 3'-isomer) Psite may be associated with 3' + 2' transacylation.

763

(e.g.,

CCA-AA) bond forming

that translocation from the A-site

to

the step

of to

(ll),

Vol.

56,

No.

3, 1974

INHIBITOR

BIOCHEMICAL

CONC.

Figure

2.

and if

2'-O-aminoacyl-tRNA

competition aminoacyl indeed

between

case.

inhibitors

of

the

containing

system

BlOPHYSlCAi

aw

(Ml

RESEARCH

INHIBITOR

COMMUNICATIONS

CONC.

(M)

Ac-(3H)Phe-puromycin formation as a function of 2'- and 3'-O-Lphenylalanyldinucleoside phosphate concentrations. The reaction mixtures were identical to those described in Figure 1 except that 1.0 X 10-4 M puromycin was also present. Compounds IIa, IIb, IIIa and IIIb were present at the concentrations indicated on the abscissa and ribosomes were added last to initiate the reactions. After 30 min at 37' the reactions were terminated (with 0.1 ml of 0.1 M Be(NO3)2 and 0.3 M NaOAc (pH 5.5) saturated with MgS04) and Ac-(3H)Phe-puromycin was extracted with 1.5 ml of ethyl acetate, as described by Monro et al. (14). The radioactivity of 1.0 ml aliquots of the ethyl acetate extract was measured in 10 ml of 4.5gPPO/lOO mg dimethyl-POPOP/0.25 liters 2-methoxyethanol/l liter Control experiments, in which toluene scintillation mixture. puromycin was omitted, indicated that little or no radioactivity Percent of Ac-(3H)Phe-puromycin was extracted to ethyl acetate. formed represents the amount of Ac-(3H)Phe-puromycin formed in the presence of aminoacyldinucleoside phosphate relative to the 100% Ac-(3H)Phe-puromycin formed amount formed in its absence. was equal to 1525 cpm. [;',I--, C-APhe (I); A, C-A(2'Phe)H (IIa); 0, C-A(2'H)Phe (IIIa). --, C-APhe (I); q , C-A(2'Phe)Me (IIb); D, C-A(2'Me)Phe (IIIb).

can be bound

puromycin,

derivatives, the

AND

the

peptidyl

P'-O-aminoacyl transferase

that

be able

acceptor

Ac-Phe

transfer

poly(U),

764

2'-o-

This

was

were

excellent

to puromycin

and 70 S ribosomes

2'(3')-0-aminoacylnucleosides compete with puromycin

the

and the

(IIb).*

IIa and IIb,

derivatives,

as donor,

to demonstrate

substrate,

and C-A(2'Phe)Me

mediated

Ac-Phe-tRNA

2 We had previously shown 2'(3')-O-aminoacyloli onucleotides transferase activity 9 12).

standard

(IIa)

C-A(2'Phe)H Both

we should

and in peptidyl

in

Vol.

56,

No.

3. 1974

BIOCHEMICAL

AN5

BlOPHYSlCAi

PUROMYCIN

Figure

Their

2).

that the

2'-

activity,

(IIIa)

C-A(2'H)Phe

good acceptor

can be seen inhibitory

from

be recognized

binding

2.5

X 10-5

the

inhibitory

(50% inhibition

In addition,

Fig. effect

The above by the

at

3, increasing of

up to the

IIa and IIb,

1.4

results peptidyl

3 Recently, C-A was found to ribosomes (13).

indicate

that

transferase

was similar

probably

as either

of the

3'-isomers

X 10m4 M) and C-A(2'Me)Phe these

compounds

to be completely

of

not

that act

inac-

at the

As

overcomes

puromycin

and the

same ribo-

derivatives

We therefore

are

shown).3

puromycin

2'-O-aminoacyl

to be a weak inhibitor

to

can exist

10m3 M (data

A-site.

765

IIb,

although

indicating

IIa and IIb,

Ac-Phe-puromycin

activity

concentration thus

of

M) which

C-A was found

at a concentration

2'-O-aminoacyloligonucleotides, site.

at

at 8 X 10 -5 M), was lower

substrates.

as an inhibitor

50% inhibition

Significantly,

(50% inhibition

somal

CONC. (M)

and 3.3 X 10m5 M for

IIa

(50% inhibition

or 3'-isomer. 2),

(IIIb)

the

(I)

of C-APhe

(Fig.

tive

inhibitory

at 4 X 10V5 M for

formation

COMMUNKATIONS

Reversal of C-A(2'Phe)H (IIa) and C-A(P'Phe)Me (IIb) inhibition of Ac-(3H)Phe-puromycin formation in the peptidyl transferase reaction. Reactions were executed as in Figure 2 except that the concentrations of IIa and IIb were held constant (4.0 X ,lO-5 and 3.4 X 10-5 M respectively) and the concentration of puromycin varied as indicated in the figure. Percent inhibition represents the difference in counts between reaction mixtures with both puromycin and the aminoacyldinucleoside phosphate present and that containing just puromycin. 0, C-A(2'Phe)H (IIa); A, C-A(P'Phe)Me (IIb).

3.

(Fig.

RESEARCH

would

of chloramphenicol

may like

M

BIOCHEMICAL

Vol.56,No.3,1974

to suggest

that

the

same may hold

AND BlOPHYSlCAi

true

for

the

here

do not

they

do show that

RESEARCH COMMUNICATIONS

3'-terminus

of

2'-O-aminoacyl-

tRNA. Although ribosomal

2'

the + 3'

data

transacylation,

can bind

to ribosomes,

however,

that

EF-T,

the

dependent

presented

presumably

isomer

binding

to the

integrity

of

establish

A-site.

the

presence

of

2'-O-aminoacyl-tRNA It

remains

2'-O-aminoacyl-tRNA

to be proven,

is retained

during

to ribosomes.

ACKNOWLEDGEMENTS This Research

investigation

was supported

Grant

No. GM-19111-02

Medical

Sciences

and in part

Cancer

Foundation

grateful

to Dr.

evaluation

of

from

the

from

in part the

by U.S.

National

by an institutional United

Harmon McAllister

Foundation

Institute grant

of Greater

(Wayne State

University)

Public

Health of

to the Detroit. for

Service

General Michigan We are a critical

the manuscript.

REFERENCES 1. 2. 3. 4. :: 7. i: 10. 11. 12. 13. 14.

Zachau, H. G. and Feldman, H. In "Progress in Nucleic Acid Research and Molecular Biology", 4, 217 (196u. Wolfenden, R., Ranmler, D. H. and Lipmann, F. Biochemistry, 3, 329 (1964). Griffin, B. E., Jarman, M., Reese, C. B., Sulston, J. E. and Trentham, D. R. Biochemistry, 2, 3638 (1966). Nathans, D. and Neidle, A. Nature, 197, 1076 (1963). Chlidek, S., Ringer, D. and femliEka, J. Biochemistry, in ress. Ofengand, J. and Chen, C.-M. J. Biol. Chem., 247, 2049 -972 e-r . Sprinzl, M. and Cramer, F. Nature New Biol., 245, 3 (1973). Ringer, D. and Chla'dek, S. FEBS Letters, in press. Hussain, Z. and Ofengand, J. Biochem. Biophys. Res. Cornnun., a, 1143 (1973). Fraser, T. H. and Rich, A. Proc. Natl. Acad. Sci. U.S.A., 70, 2671 (1973). T. and Lessard, J. L. J. Biol. Chem., 245, 6208 Pestka, S., Hishizawa, (1970). Rychlfk, I., Cernd, J., Chla'dek, S., Pulkrabek, J., Zemlizka, J. and Haladovd, Z. FEBS Symposium, 2l-, 47 (1970). Nierhaus, D. and Nierhaus, K. H. Proc. Natl. Acad. Sci. U.S.A., 70, 2224 (1973). J. and Marcker, K. A. Proc. Natl. Acad. Sci. U.S.A., Monro, R. E., terni, 6J, 1042 (1968).

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