Inhibition of aminoacyl-sRNA binding to ribosomes by tetracycline

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INHIBITION OF AMINOACYL-sRNABINDING TO RIBOSOMJZS BY TETRACYCLINE Gerard0 Suarez* and Daniel Nathans Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Md.

ReceivedJanuary15,

1965

Tetracycline

antibiotics

(Hash, 1963) and in extracts Ochoa, 1962; Franklin, the tetracycline5 cation

inhibit

of bacterial

1963).

binding

complex, and peptide

(Rendi and

pathway beyond the esterifi-

to distinguish peptide

three steps in the trans-

linkage:

binding

of messenger RNA

of aminoacyl-sRNA to the messenger RNA-ribosome We report

bond formation.

contrast

to chloramphenicol

specific

binding

Similar

in bacteria

and mansnalian cells

biosynthetic

is now possible

fer of amino acids from sRNA into to ribosomes,

synthesis

In cormnonwith puromycin and chloramphenicol

block the protein

of sRNA. It

protein

here that tetracycline,

and puromycin (Spyrides,

1964), inhibits

in the

of aminoacyl-SRNA to the ribosome-messenger RNA complex.

observations

have been made independently

by Hierowski

and Lipmann

(1964). Materials Preparation

of E. &&

and Methods

B, washed ribosomes,

high speed supernate

(S105) and esterified

sRNA were carried

out as previously

and Lipmann, 1961).

C14-phenylalanyl-sR

was acetylated

Haenni and Chappeville to publication. lysed with

0.1 N aassonia, all

Binding

to whom we are grateful

When the resulting

N-acetyl-phenylalanine (4:l:S).

(1964),

described

by the method of

for information

Cl4 -N-acetyl-phenylalsnyl-sRNA

the radioactivity

migrated with

termined by the method of Nirenberg

and.Leder

(1964).

was hydro-

acid-water

to ribosomes was deC14-polyuridylate

* Rockefeller Foundation Fellow, on leave from the Institute Chemistry, University of Chile, Santiago, Chile. 743

prior

reference

on paper chromatography in butanol-acetic

of Cl4 -N-acetyl-phenylalanyl-sRNA

(Nathans

of Physiological

Vol. 18, No. 5-6, 1965

was prepared Schwarz lase

by polymerization

Bioresearch

from_M.

product

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

Orangeburg,

lvsodeikticus

was treated

sephadex.

Inc.,

with

Radioactivity

of Cl4 UDP (21 mC/m Mole,

(a gift bentonite

N.Y.) of Dr.

was measured

sRNA was purchased

from General

was supplied

U),

from Miles

by Chas.

Pfizer

Roland

F. Beers,

on filter

based on the procedure

(poly

polynucleotide Jr.).

and Co.,

discs

Co.,

Co.,

Chagrin

Clifton,

New York,

The through

625

in a toluene-

of Mans and Novelli

Biochemicals Chemical

paper

from

phosphory-

to remove enzyme and passed

based scintillator

uridylate

with

purchased

(1960).

Falls,

N.J.

Ohio;

4. & poly-

Tetracycline

New York.

Result% As reported transfer (figure

of amino acids 1) .

by others from

Under conditions

(Rendi

and Ochoa,

sRNA to polypeptide of

1 5

limiting

18

ribosome

15

1962; is

Franklin,

inhibited

1963)

the

by tetracycline

and phenylalanyl-sRNA

20

TETRACVCLIWE, M x la5

i?,dlmm 1:

Inhibition by tetracycline of polyphenylalmine synthesis from phanylalanyl-si?NA. The following were incubated in a volume of 0.11 ml: 0.05 n Trir Bcl pH 7.4, 0.10 Y El, 0.014 x n&12, 0.01 M glutathioaa, 0.01 Bl phoaphoanolpyruvate, 2 pg pyruvate kinaae, 0.0001 H GTP, 0.5 m poly U, 0.48 A260 units of ribosoma, 5 pL 5105 md 50 pg slUU charged with C14-phenylalmine (8.5 x 104 cts/ud.n/w rRRA; 3.0 x 10s cts/d.d~ After incubation foe 10 min. at 3ooC, Mole phenylalanina). 0.1 ml aliquots were transferred to filter paper discs and analysed for protein radioactivity as notad in methods. In the absence of tetracycline 900 cpm was found in polypeptide. 744

Vol. 18, No. 5-6,1965

concentrations, alanine

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

inhibition

was detectable

of incorporation at a tetracycline

As shown in Table 1, partial

reversal

of phenylalanine level

into

polyphenyl-

of 10m7 M and maximal at 10m4 M.

of inhibition

was observed at increased

TABLE 1

Jncorrboratiop Ribosoum 460 id&&

sJid&Jl

-Tet

us!a& Inhibitioq

ix!&

0.56

0.2

440

176

60

1.4

0.5

1400

838

40

2.8

1.0

2240

1690

25

7.0

2.5

2220

2060

6.5

Reversal of-the

tetracvcline

effect

by jncreased

and oolv U.

ribosoms

Incubation conditions are given in the legend of figure 1, except that the volume was 0.22 ml, the concentrations of ribosomes and poly U varied as noted in the table, and 100 ug sRNA (SSOO et&sin) was present. Tetracycline was added (+ Tet) at a concentration of 5 x 1~6 M.

ribosome plus poly U concentrations. increased

separately

consistent

When each of these components was

the decrease in inhibition

than when both were increased

of inhibition affected

was less marked and less

together.

In contrast

by ribosomes plus poly U, the degree of inhibition

by an eight-fold

by a five-fold

increase

The effect

increase

in phenylalanyl-sRNA

in the supernatant

of tetracycline

on the binding

et al.,

1963; Spyrides,

We have confirs&

1964; Raji and Raji,

ribosomes,

poly U and Cl40 phenylalanyl-sRWA,

negligible

hot trlchloroacetic

acid-precipitable 745

concentration

nor

of C14-phenylalanyl-sR initially

the ribosomes at 105,000 g for 2 hrs. and more recently and Leder (1964).

was not

fraction.

to ribosomes in the presence of poly U was studied

Nirenberg

to the reversal

by the method of

the results 1964) that

by sedimenting

of others

incubation

under conditions radioactivity

(Nakamoto of washed

where is detectable,

Vol. 18, No. 5-6, 1965

results

8lOCHEMlCAL AND 8lOPHYSlCAL RESEARCH COMMUNICATIONS

in ribosome-associated

exclude the possibility

that

counts, acid-soluble

somes under these conditions, to study the binding

fiable

after

hydrolysis

chromatography.

to the lack of effect

After

incubation

the radioactivity

were formed on the ribo-

of this

material

with

bound to ribosomes was identi-

in 0.1 N anrnonia as N-acetyl-phenylalanine

Conditions

As shown in the table,

peptides

In order to

we have used C14-N-acetyl-phenylalanyl-sRNA

reaction.

ribosomes and poly U all

dependent on poly U.

for the binding

tetracycline

inhibits

reaction

by co-

are given in Table 2.

the binding

reaction,

in contrast

of puromycin and chloramphenicol. TABLE 2

Fte/m inbound

Conditions

&9 ribosomes

Complete system

1700

-Poly u

310

-Ribosomes

156

+Puromycin low3 M

1670

tChloramphenico1

1610

iTetracycline

4 x lo-4 M

10'4 M

940

a.binding a N-acetvl-Rhenvlalanvl-sRNA u riboaomea The following were incubated in a volume of 0.10 ml: 0.05 M Tria UC1 pH 714, 0.02 MMgCl2, 40 wg poly U, 4.4 A260 units of ribosomes, 0.10 M RCl, 0.01 M glutathione, and 92 pg sRNA eaterified with C 14-N-acetyl-phenylalanine (4.2 x 104 cta/min/mg sRNA; 3.0 x 108 cts/min/p Mole N-acetyl-phenylalanine). After incubation at 300 for 10 min. 6 ml. of cold buffer was added (Trie HCl 0.1 M pH 7.2, MgC12 0.02 M, lCC1 0.1 M) and ribosomes collected, washed and counted on a Millipore filter as described by Nirenberg and Leder (1964).

The effect binding

of different

concentrations

of Cl4 -N-acetyl-phenylalanyl-sRNA

A comparison of the results to tetracycline formation.

Although

this

2.

1 and 2 suggests that the sensitivity

reaction

difference

on the

to ribosomes is shown in figure

of figures

of the binding

of tetracycline

is less than that of polypeptide

is due in part to the different 746

ribosome

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 18, No. 5-6, 1965

M x lo5

TETRACYCLIWE, Finure

Inhibition by tetracycline of the binding of N-acetylphenylalanyl-sRNA to ribosomes. Incubation conditions given in the legend of Table 2.

2:

and poly

U concentrations

difference

in maximal

is

almost

about

completely

50 per cent Since

template

the

cycline poly

inhibition inhibited,

the binding

tetracycline

effect

3).

of

is

is

a striking

polypeptide

inhibited

That

in the

this

is not

to the

synthesis extent

of

to the

the binding

on a polynucleotide

presented absence

of tetracycline

the effect

of messenger

of the

of tetrarecovered

and 80 per cent

conditions,

was inhibited

binding

42 per cent

of tetracycline

ribosome-messenger

747

and absence

83 per cent

Under similar

that

of the binding

the case was shown by measuring

in the presence

to ribosomes

We conclude

with

depends

be due to inhibition

10m4 M tetracycline.

of aminoacyl-sRNA

due to interference

could

In the experiment

M tetracycline.

the binding

binding

U to ribosomes

of C14-N-acetyl-phenylalanyl-siINA by 10’4

there

two reactions:

of aminoacyl-sRNA

U was bound to ribosomes

in the presence

of these whereas

to ribosomes.

of Cl4 -poly

(figure

incubations,

only.

of polynucleotide the binding

needed in these

are

RNA complex

RNA to ribosomes.

on is not

Vol. 18, No. 5-6, 1965

5

Finure

9:

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

10

15

25

25

5

FRncllOR

NO.

10

15

20

25

Lack of effect of tetracycline on the binding of poly U to ribosomes. The following were incubated in a final volm of 0.2 ml: 0.05 M Tris UC1 pH 7.4, 0.01 H Mg acetate, 0.10 M KCl, 6 ug Cl4 poly U (6.5 x 106 ctsfminhg , 14 A260 units of ribosomes, and where indicated below, 10' 1 M tetracycline. All components except poly U were incubated for 10 minutes at 300, after which poly U was added, and the adxture allowed to remain at Ooc for 20 min. Centrifugation was carried out in an SW 39 Spinco rotor at 35,000 rpm for 90 min. in 5 ml of a linear 5 to 20 per cent sucrose gradient containing 0.01 M Tris UC1 pH 7.4, 0.01 M Mg acetate and 0.10 M KCl. Three-drop fractions were collected from the bottom of each tube and 0.05 ml taken for determination of cold trichloroacetic acid-precipitable radioactivity (-). A260 was measured at a dilution of 1:7 (..... ). A, complete system without tetracycline; B, complete system with 10'4 M tetracycline present during incubation and in the gradient. Centrifugation of Cl4-poly U in the absence of ribosomes gave a single peak of radioactivity at tube number 23.

Discussion The inhibitory

effect

of tetracycline

to be due to inhibition

of binding

messenger BNA complex.

Rather extensive

acids and to proteins, (Kohn, 1961); this hibitory

effect

polyphenylalanine

in protein synthesis

synthesis.

synthesis

appears

of aminoacyl-sRNA to the ribosomebinding

mediated by divalent

type of binding

on protein

of tetracycline

cations,

at a critical

has been reported

site

The observations

by tetracycline

is partially

creased ribosomes and poly U but not by increased 748

to nucleic

may underlie that

its

inhibition

inof

Overcome by in-

aminoacyl-sRNA suggests

Vol. 18, No. 5-6, 1965

that

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMJNICATIONS

the antibiotic

may bind to one or more sensitive

concerned with binding that

of sRNA (Gilbert,

there are two binding

Rich,

1964; Arlinghaus

sites

1963).

reaction

antibiotic

only to the extent

Since tetracycline

the attachment

of polypeptitie

formation

of binding

of successive

molecules

it

inhibits

is possible

the that the

only one of the two sites

of sRNA. Alternatively,

may simply

reflect

the partial

of aminoacyl-sRNA during

phenylalanyl-sRNA.

blocked the synthesis This inhibition

plus poly U in the incubation or supernatant

fraction.

and chloramphenicol, alanyl-sRNA unaffected. completely

the greater inhibition

growth of the

Although

from

ribosomes

mixture

but not by increased

minoacyl-sRNA

In contrast

to the lack of effect

of puromycin

tetracycline

to ribosomes,

of polyphenylalanine

was reversed by increased

reduced the binding

whereas the binding

polyphenylalanine

by tetracycline, about 50 per cent.

of N-acetyi-phenyl-

of poly U to ribosomes was

synthesis

aminoacyl-sRNA binding

was inhibited

almost

to ribosomes was inhibited

We suggest that tetracykline

ribosomes in such a way as to obstruct

one of the two sites

may combine with for the binding

of aminoacyl-sRNA.

This research has been supported by grant No. GM 10180 from the National Institutes

on

chain.

Tetracycline

maximally

of 50 per cent,

binds in such a way as to obstruct

inhibition

peptide

Recent work has indicated

1964), one of which may hold the peptidyl-sRNA

et al.,

the ribosome concerned with

on the ribosome

for sRNA on each 70s ribosome (Warner ancl

and the other the next sminoacyl-sRNA. binding

sites

of Bealth,

USPES. 749

Vol. 18, No. 5-6, 1965

BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Arlinghaus, R., Shaeffer, J. and Schweet, R., Proc. Nat. Acad. Sci., Wash., 2, 1291 (1964). Franklin, T. J., Biochem. J., 87, 449 (1963). Gilbert, W., J. Mol. Biol., 5, 389 (1963). Haenni, A. L. and Chappeville, F., J. Mol. Biol., in press. Hash, J. H., Fed. Proc., z, 301 (1963). Hierowski, M. and Lipmann, F., Proc. Nat. Acad. Sci., Wash., in press. Kaji, A. and Kaji, H., Biochem. Biophys. Res. Corn., I& 186 (1963). Kohn, K. W., Nature, 191, 1156 (1961). Mans, R. J. and Novelli, G. D., Arch. Biochem. 94, 48 (1961). Nakamoto, T., Conway, T. W., Allende, J. E., Spyrides, G. J. and Lipmann, 'F., Cold Spring Harbor Symp. Quent. Biol. 2, 227 (1963). Nathans, D. and Lipmann, F., Proc. Nat. Acad. Sci., Wash., 41, 497 (1961) Nirenberg, M. and Leder, P., Science 145, 1399 (1964). Rendi, R. and Ochoa, S., J. Biol. Chem., 273, 3711 (1962). Spyrides, G., Proc. Nat. Acad. Sci., Wash., z, 1220 (1964). Warner, J. R. and Rich, A., Proc. Nat. Acad. Sci., Wash., x, 1134 (1964)

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