action of tetracycline and puromycin

8/19/2019 Action of Tetracycline and Puromycin

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THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 247, No. 1, Issue of January 10, PP. 45-50, 1972

Printed in U.S .A.

Studies on the Action of Tetracycline and Puromycin*

(Received for publication, June 14,

1971)

SHIGEAKI TANAKA, KAZUEI

IGARASHI, AND

AKIRA

KAJI

From the Department

of

Microbiology School

of

ikfedicine University

of

Pennsylvania Philadelphia Penn-

sylvania

i91 O/t

SUMMARY

By means of a convenient method for separating phenyl-

alanyl puromycin f rom diphenylalanyl puromycin, it was

found that, upon reaction with puromycin, phenylalanyl

transfer RNA bound to ribosomes at5 to 6 mu Mg++ yields

phenylalanyl puromycin exclusively while phenylalanyl-

tRNA bound at 13 mu Mg++ yields diphenylalanyl puromycin

as well as phenylalanyl puromycin. Tetracycline inhibited

mostly the binding of phenylalanyl-tRNA to the acceptor site,

but the binding to the donor site may be inhibited at 13 mu

Mg++. Puromycin reaction of phenylalanyl-tRNA bound

to the donor site was inhibited by the presence of N-ace-

tylphenylalanyl-tRNA at the acceptor site.

It has been suggested that there are two ribosomal sites for the

binding of aminoacyl transfer RNA (l-8). One of the two sites

is sensitive to puromycin and has been called the donor site (site

2) (5, 7). The other site has been called the acceptor site (site 1)

(5, 7). In the presence o f low concentrations of Mg++ (5 to 6

mM), the bound phenylalanyl-tR.NA reacted with puromycin in

the absence of G facto r; in the presence of relatively high Mg++

(13 mM), two ribosomal sites were presumably occupied by

phenylalanyl-tRNA (1). These considerations provided a con-

venient method of assaying the action of antibiotics such

as erythromycin, lincomycin, and streptomycin (9).

In this communication, we extended our studies on the ribo-

somal sites with the method which separates phenylalanyl puro-

mycin from diphenylalanyl puromycin to elucidate the action of

tetracycline and puromycin. Concerning the mode of action of

tetracycl ine, it is believed that tetracycline inhibits the binding

of aminoacyl-tRNA to the acceptor site of the ribosomes (10-12).

However, results of the NH?-terminal analysis of polyphenyl-

alanine formed from the complex which was prepared in the

presence of tetracycline suggested that the binding of aminoacyl-

tRNA to the donor site may also be influenced (7). The availa-

bility o f the method to determine phenylalanyl puromycin as

well as diphenylalanyl puromycin quantitat ively made it possible

to measure the percentage of inhibition of the binding of phenyl-

alanyl-tRNA to the acceptor and the donor site, respectively.

* This research was supported by United States Public Health

Service Grant GM-12,053, National Science Foundation Grant

GB-18355, and Damon Runyon Memorial Fund for Cancer Re-

search, Grant 799-DT.

It was found that tetracycline inhibits not only the acceptor site

binding but also the donor site binding of phenylalanyl-tRNA in

the presence of high Mg* (13 ma/r).

The reaction of puromycin with aminoacyl-tRNA on the ribo-

some has been used extensively for studying peptide bond forma-

tion. However, it has not been clear whether puromycin ap-

proaches the donor site from the side of the acceptor site or not.

By use of the complex having N-acetylphenylalanyl-tRNA both

on the acceptor and the donor site, we concluded that puromycin

approaches the donor site from the side of the acceptor site.

MATERIALS AND METHODS

Escherickia co& Extract and Other hlaterials-Preparation of

ribosomes, tRNA from E. coli B, and aminoacyl-tRNA have been

described in the previous communications (1). The ribosomes

were washed three times with a buf fer containing 0.1

M

Tris-HCl

(pH 7.8), 0.01

M

magnesium acetate, 0.06

M

potassium chloride,

0.006

M

P-mercaptoethanol, and 0.5

M

ammonium chloride, and

were free from aminoacyl-tRNA transfer factor (T factor) and

initiation facto rs. Preparation of T factor and G factor was as

described previously (13).

Binding o f [‘4~Phenylalanyl-tRNA to Ribosomes

and

Isolation

of Complex of Ribosmes, poZy(U), and [‘4C]Phenylalanyl-tRNA-

A typical binding reaction mixture (0.5 ml) for the isolation of the

complex, unless otherwise specified, contained 50 mM Tris-HCl

(pH 7.2), 6 mM magnesium acetate, 40 mM ammonium chloride,

400 pg of poly(U), 5.2 mg of tRNA containing 8.5 X lo5 cpm of

[14C]phenylalanyl-tRNA, and 5.8 mg of ribosomes. This binding

condition was called condition A. In some cases, the binding

reaction was performed under identical conditions except that

the Mg+f concentrat ion was 13 mM. This was called condition

B. When the enzymatic binding of aminoacyl-tRNA

was

carried

out, 85 pg of T fac tor , 0.2 mM GTP, and 2 ml4 dithiothreitol were

also added to the above binding mixture. The reaction mixture

was incubated for 20 min at 22”. To separate the complex of

ribosomes, poly(U), and [i4C]phenylalanyl-tRNA from unbound

[‘%]phenylalanyl-tRNA, the mixture was placed on 5 ml of a

linear sucrose gradient solution (5 to 20’%) containing 20 mM

Tris-HCl (pH 7.2), 6 mM or 13 mM magnesium acetate, 40 mM

NH&I, and 6 mM P-mercaptoethanol. The gradients were cen-

trifuged for 65 min at 48,000 rpm in a Beckman-Spinco rotor (SW

50.1), and the 70 S ribosome portions were collected as the ribo-

somal complex.

Reaction of Bound [14C]Phenylalanyl-tRNA with Puromycin-

The reaction mixture (0.5 ml) for the puromycin reaction con-

tained 20 mM Tris-HCl (pH 7.2), 13 mM magnesium acetate, 80

mM NH&l, 0.2 mM GTP, 24 pg of G factor , 1 mM puromycin, and

45

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46

Action of Tetracycline and Puromycin Vol. 247, No. 1

0.3 ml of the fraction containing the ribosomal complex isolated

as described above. The reaction mixture was incubated for 60

min at 22”, mixed with 2 ml of 0.01

M

Tris-HCl (pH 7.8), and

shaken well with 6 ml of ethyl acetate.

The ethyl acetate extract

(0.5 ml) was mixed with 5 ml of Bray’s solution (14), and the

radioactivity was measured by liquid scintillation counter. The

ethyl acetate extract (5 ml) wa,s evaporated in a vacuum, redis-

solved in 0.6 ml of 0.5

M

acetic acid, and applied on a Sephadex

G-15 column as described below. Approximately 70 to 75% of

the bound [14C]phenylalanyl-tRNA reacted with puromycin under

these conditions.

Separation of Phenylalanyl Puromycin and Diphenylalanyl

Puromycin by Xephadex G-15 Column Chrmatoglaphy-The pu-

romycin derivat ives of phenylalanine formed as described in the

preceding section were analyzed by Sephadex G-15 column chro-

matography (15). The puromycin derivat ive in 0.5 ml of 0.5

M

acetic acid was applied to a Sephadex G-15 column (0.8 x 150 cm)

which had been equilibrated with 0.5

M

acetic acid. The elution

was carried out by 0.5

M

acetic acid with a flow rate of 1 ml per

12 to 15 min. Each fract ion (1 ml) was collected and mixed with

5 ml of Bray’s solution and the radioactivity of the [14C]phenyl-

alanyl puromycin derivat ives was measured.

The elution profi le

is shown in Fig. 1.

IdentQication of Purcnnycin Deriva tives of Phenylalanine-To

identify the nature of two peaks of [%]phenylalanyl puromycin

derivat ives (Fig. l), NHz-terminal analysis of each peak was per-

formed. Upon dinitrophenylation, [14C]phenylalanyl puromycin

would give dinitrophenyl [14C]phenylalanyl puromycin which

would be hydrolyzed by 6

N

HCl into dinitrophenyl [14C]phenyl-

alanine and puromycin. The dinitrophenyl phenylalanine is

soluble in ether. Thus, under these procedures, lOO’% of radio-

act ivi ty should become ether soluble.

On the other hand, under

identical conditions [14C]diphenylalanyl puromycin would yield

only 50% of its radioactivity as ether-soluble material because of

insolubility of phenylalanine into ether. The materials of the

first peak (Fractions 38 to 42 in Fig. 1, A and B) and the second

peak (Fractions 48 to 58 in Fig. 1B) were separately pooled,

dried in a vacuum, and dissolved in 0.2 ml of 1 Zo riethylamine.

Dinitrophenylation was started by addition of 0.2 ml of 5%

solution of dinitrofluorobenzene in alcohol and carried out for 4

hours at room temperature. After the dinitrophenylation was

completed, 6

N

HCl (0.2 ml) was added to the reaction mixture,

and free dinitrophenol was removed by ether extraction. Under

these acidic conditions, most of the dinitrophenyl [14C]phenylal-

any1 puromycin derivat ives remained in the aqueous layer. The

concentration of HCl of these aqueous layers was brought to 6

N

by the addition of concentrated HCl, and acid hydrolysis was

performed at 110’ for 6 hours. The resulting hydrolysate was

diluted to 0.6

N

HCl with water, and ether-soluble materials ex-

tracted three times with 5-ml portions of ether. The radioac-

tivities in the combined ether extracts and aqueous layer were

compared. With material of the first peak, a major portion

(about 90%) o f its radioactivity was converted to ether-soluble

material indicating that the first peak represents phenylalanyl

puromycin. When the second peak was subjected to the identi-

cal NH*-terminal analysis, about 53yo of the radioactivity was

converted to the ether-soluble material showing that the second

peak represented diphenylalanyl puromycin.

JJateriaZs-Preparation of N-acetylphenylalanyl-tRNA was

performed as described previously (16). Puromycin hydrochlo-

ride was purchased from Nutritional Biochemicals, and tetracy-

cline hydrochloride was from California Biochemical Co. Speci-

fic act ivi ty of [14C]phenylalanine was 375 &i per pmole and the

counting eff iciency was lo6 cpm per PCi.

RESULTS

Characterization of

Complex

Formed

under

Conditions A and B-

It has been postulated that in the presence of 5 to 6 mM Mg++

concentration, phenylalanyl-tRNA binds to the donor site alone,

and in the presence of 13 my Mg+f concentration, both donor

and a.cceptor sites are occupied. Our previous studies, on the

mode of action of antibiotics (9), were dependent on this assump-

tion. It was therefore desirable to obtain the evidence which

would indicate that this is indeed the case. From this assump-

tion it follows that we should have mostl y phenylalanyl puromy-

tin when the binding was carried out at low Mgf+ (condition A)

while diphenylalanyl puromycin as well as phenylalanyl puromy-

tin would be formed when the binding was carried out at high

Mg* (condition B).

Fig. 1A shows that the puromycin derivat ive formed under

condition A gives one peak corresponding to a phenylalanyl pu-

romycin upon passage through the Sephadex G-15 column. As

expected from the function of the G factor, the addition of the

factor did not influence the nature or the quantity of the product

as shown in this figure. On the other hand, when the complex

was made under condition B, the addition of the G factor and

GTP caused a large increase o f formation of phenylalanyl puro-

mycin and diphenylalanyl puromycin as shown in Fig. 1B.

Eff ect of Tetracycline on Binding of Phenylalanyl-tRNA--It has

been proposed that tetracycline has an exclusive inhibitory effect

on the binding of aminoacyl-tRNA to the acceptor site (l&12).

On the other hand, during the studies on the relationship between

the acceptor site and the donor site, it was observed that, under

certain conditions, tetracycline may also inhibit the binding of

phenylalanyl-tRNA to the donor site (7). I t was therefore

desirable to determine exact ly the percentage of inhibition of the

binding of aminoacyl-tRNA to each site.

In the experiment shown in Table I the ribosomal complex was

prepared under various conditions in the presence or absence of

tetracycl ine. The puromycin derivatives o f [lJC]phenylaline

were formed f rom these complexes and analyzed with Sephadex

G-15 column. It should be pointed out that the inhibitory ef fec t

of tetracycline on the binding reaction was much more pro-

nounced when the binding reaction was carried out in the pres-

ence of high Mg++ or T factor .

In order to determine which site was more susceptible to

tetracycline, the formation of puromycin derivatives of the

bound phenylalanyl-tRNA was studied with or without G facto r.

AS shown in Table I the addition of G factor stimulated the

formation of phenylalanyl puromycin derivat ives when the

ribosomal complex prepared at high Mgf+ (13 mM) or in the

presence of T factor was used. Table I also shows the results of

the analysis o f the puromycin derivatives formed from these

complexes. The relative amounts of phenylalanyl puromycin and

diphenylalanyl puromycin were calculated. With these values,

it was possible to estimate the distribution of phenylalanyl-

tRNA reactive with puromycin between the donor and the

acceptor site. The principles used in calculating the distribution

of phenylalanyl-tRNA between the two sites are (a) phenylalanyl

puromycin formed in the absence of G factor was assumed to be

located at the donor site; (b) diphenylalanyl puromycin was

derived from phenylalanyl-tRNA bound to two sites; thus the

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Issue of January 10, 1972 X. Tanaka, K. Igarashi, and A. Kaj i 47

I

IO

20 30 40 50 60

1

I

70

IO

I

20 30 40 50 60 70

Fraction Number

FIQ.

1 (lef t and center). Sephadex G-15 column chromatography

of puromycin derivat ives of [~4C]phenylalanine. The puromycin

derivat ives of [“C]phenylalanine prepared under Conditions A

(5,520 cpm) or B (20,292 cpm) were extracted in ethylacetate,

the ethylacetate was evaporated and dissolved in 0.6 ml of 0.5

M acetic acid. The solution was then poured onto a column

(0.8 X 150 cm) of Sephadex G-15 which had been equilibrated

with 0.5 M acetic acid. The sample was eluted with 0.5

M

acetic

acid with flow rate of 1 ml eve ry 12 to 15 min.

Fractions (1 ml)

were collected and l-ml aliquot was mixed with 5 ml of Bray’s

solution (14) and the radioactivity was counted. O-0, puro-

mycin derivat ives formed in the presence o f G factor; O- - -0,

puromycin derivat ives formed in the absence of, G facto r.

A

and

B

represent puromycin derivat ives formed under Conditions A

and B, respectively.

FIG. 2.

(right). Ef fec t of additional binding of aminoacyl-tRNA

on the puromycin reaction of the donor site-bound N-acetyl[ iV]-

phenylalanyl-tRNA. For the preparation of the ribosomal

complex having N-acetyl[14C]phenylalanyl-tRNA mostl y at the

donor site, the binding reaction mixture (0.4 ml) contained 40

Fraction Number

lncubatlon trne lmin)

mu Tris-KC1 (pH 7.2), 40 rnM NH&l, 5 mM magnesium acetate,

80 fig of poly(U), 2.5 mg of ribosomes, and 42 fig of tRNA mixture

containing 2 X 10” cpm of N-acetyl[14C]phenylalanyl- tRNA.

After incubation for 15 min at 22’, 2.3 mg of tRNA mixture con-

taining 1.2 mpmoles of N-acetyl[laC]phenylalanyl-tRNA or 3.0

mg of tRNA mixture containing 1.5 voles of [‘*C]phenylalanyl

tRNA were added to the above binding reaction mixture and

Mg++ was adjusted to 13 mM. The mixtures were incubated

again for 15 min at 22’ to assure the additional binding to the

acceptor site. The ribosomal complex thus prepared was isolated

as described in the text .

The reaction mixture for the puromycin

reaction (0.5 ml) of the bound N-acetyl(14C]phenylalanyl-tRNA

was as described in the text and it contained approximately 2,266

cpm of bound N-acetyl[l*C]phenylalanyl-tRNA, but no G factor.

N-Acetyl[i4C]phenylalanyl-pyromycin formed is expressed as

percentage of the total bound iV-acetyl[14C]phenylalanyl-tRNA.

O-O, complex with no additional [l%]aminoacyl-tRNA at

the acceptor site; A-A, complex with N-acetyl[i%]phenyl-

alanyl-tRNA at the acceptor site; H, complex with addi-

tional [%]phenylalanyl-tRNA at the acceptor site.

TABLE

I

Effects

of tetracycline on nonenzymatic and enzymatic

binding

of

[“C]phenylalanyl-tRNA to ribosomes and

puramycin reaction

of bound

[W]phenylalanyl-tRNA

The reaction mixture for the preparation of the ribosomal com- mation o f puromycin derivat ives of [%]phenylalanine, and the

plex was described in the text except that it contained 4 mg of ribo-

column chromatography of the puromycin derivatives were car-

somes, 32Opg o f poly(U), and 1.8 X 106 cpm of [14C]phenylalanyl-

ried out as described in the text. Where indicated 15 ag of G

tRNA (4 mg of tRNA); in some cases, 85rg of T factor, 2 mM dithi-

facto r was added to the reaction mixture for the formation of

pure-

othreitol, and 0.2 mu GTP were added. The isolation of the

mycin derivatives.

complex of [14C]phenylalanyl- tRNA, poly(U), and ribosomes, for-

Binding condition of [W]phenylalmyl-tRNA

t&w

6

6

6

6

13

13

T factor

-

__

-

Tetracycline

- I’

-

M

CM

0

8,850

5 x 10-d 7,500

0 12,600

5 x 10-d 8,580

0 17,580

5 x 10-d 10,430

-

I

Bound

~W)phtmylalanyl-tRNA

-

I

.-

-_

Total phenylalanyl puromycin formed

G factor

-

+

-

+

-

+

+

-

+

-

+

-

CPm

Phenylalanyl

puromycin

Diphenylalanyl

puromycin

6,190

92

8

6,640

89

11

6,020

92

8

6,020

90

10

7,680

64

36

11,110 58 42

6,100 96 4

6,160 94

6

5,300 49 51

11,400 47 53

3,890 94

6

4,930 91 9

-

I

Puromycin derivatives formed

(percentage of total)

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48

Action of Tetracycline and Puromycin

Vol. 247, No. 1

T ABLE

II

Tetracycline inhibition of binding of phenylalanyl-tRNA”

The calculations of the amount of the puromycin-reactive [‘*Cl-

phenylalanyl-tRNA at the acceptor and the donor site were car-

ried out as follows. For example, f rom the ribosomal complex

formed in the presence of 6 mM Mg*, 6190 cpm of the puromycin

derivat ives of [lQZ]phenylalanine were formed in the absence of

the G factor (line 1 in Table I). Of this, 92yo (5695 cpm) were

phenylalanyl puromycin and 8% (495 cpm) were diphenylalanyl

puromycin. Since diphenylalanyl puromycin must come from

the phenylalanyl-tRNA at the donor and the acceptor site, 248

cpm (495 X . = 248) were assigned to each of these sites. The

phenylalanyl puromycin (5695 cpm) must come entirely from the

donor site. In the presence of the G facto r, 6640 cpm of puro-

mycin derivatives of [r%]phenylalanine were formed (line 2 in

Table I). Of this, 89% (5910 cpm) and 11% (730 cpm) were

[‘4C]phenylalanyl puromycin and diphenylalanyl puromycin, re-

spectively. Thus, 215 cpm (5910 - 5695 = 215) of phenylalanyl

puromycin were formed due to the addition of G factor.

We re-

gard that this was bound at the acceptor site and translocated to

the donor site by the G facto r. The diphenylalanyl puromycin

formed due to the addition of G factor was 235 cpm (730 - 495 =

235). We assign 117 cpm (235 X + = 117) to eachsite. The total

[r%]phenylalanyl-tRNA bound to the donor site is therefore

5695 + 248 + 117 = 6060 cpm and that bound to the acceptor site

is 215 + 248 + 117 = 580 cpm. The sum of these values (6060 +

580 = 6640) represents total puromycin-reactive [r%]phenpl-

alanyl-tRNA bound in the presence of 6 mM Mg++ (line 2 in Table

I).

Binding conditions

M g + +

m M

6

0

6

-

5 x 10-4

6

+ 0

6 +

5 x lo-’

13

- 0

13

- 5

x 10-d

T

factor

Tetracycline

Calculated

amount of

puromycin-reactive

[‘4C]phenylalanyl-

tRNA at each site

DOnOr

Acceptor

site

site

cpl

GO60 580

5839 181

7402 3709

6041 119

5618 5782

3878 1052

Percentage of

hnhibition of binding

,f [‘rC]phenylalanyl-

tRNA to each site

by tetracycline

DOnOr Acceptor

site site

4 69

18 97

31 1 82

a Data were derived from Table I.

radioactivity of this fraction was divided by two and equally

distributed to the acceptor and donor site; and (c) phenylalanyl

puromycin dependent on the presence of G factor was allotted

to the acceptor site. The results of these calculations are sum-

marized in Table I I.

It is clear from Table II that the ef fec t of tetracycline was

mainly on the acceptor site when the binding reaction was carried

out at 6 mM Mg++

or in the presence of T facto r.

On the other

hand, when the binding reaction was carried out at 13 mM Mg”,

the effect of tetracycline on the binding of phenylalanyl-tRNA

to the donor site became significant. However, even under these

conditions, the inhibition of the binding of phenylalanyl-tRNA

to the acceptor site was much larger than that to the donor site.

It should be noted that the assumption (a) used in calculating

these values may not necessarily be correct in view o f the fac t

that a fair amount of nonenzymatic translocation of diphenyl-

alanyl-tRNA takes place (17). If one assumes that similar

nonenzymatic translocation takes place with phenylalanyl-tRNA

bound at the acceptor site, the inhibitory eff ect of tetracycline

at the donor site becomes less than a few percentage even in the

presence o f 13 mM Mg++. In view of the fac t that approximately

equal amounts of phenylalanyl-tRNA are bound to donor and

acceptor site in the presence of 13 mM Mg++ (2), nonenzymatic

translocation of phenylalanyl-tRNA is probably not as much as

that with diphenylala,nyl-tRNA. This is because non-enzymatic

translocation of phenylalanyl-tRNA would give a far greater

amount of phenylalanyl-tRNA at the acceptor site.

At any rate,

one can conclude that a major action of the inhibitory eff ect of

tetracycline is on the acceptor site. It can also be seen in this

table that the stimulation of the binding of [r4C]phenylalanyl-

tRNA by T factor was mainly on the acceptor site in confirmation

of our previous conclusion with the experiment where NH%-

terminal analysis of polyphenylalanine was employed to deter-

mine the amount of bound phenylalanyl-tRNA at these sites (2).

Studies on Puromycin Action and Influence of Aminoacyl-

tRNA at Acceptor Site on Reac tivi ty of Aminoacyl-tRNA Bound

at Donor Site-Since the puromycin reaction with the donor site-

bound aminoacyl-tRNA is analogous to the peptide bond forma-

tion between aminoacyl-tRNAs bound at the donor and acceptor

sites (B-20), it is reasonable to assume that puromycin would

attack the aminoacyl-tRNA at the donor site from the side of

the acceptor site. If this assumption is correct, one would

expect that the presence of nonreactive aminoacyl-tRNA at the

acceptor site would hinder the reactivi ty of the aminoacyl-tRNA

at the donor site.

The report contrary to this expectation (21)

prompted us to examine this possibility with the present system.

In the experiment described in Table III , the complex of [‘“Cl-

phenylalanyl-tRNA, poly(U), and ribosomes was prepared at

5 mM Mg++. This complex was then incubated with or without

N-acetylphenylalanyl-tRNA or phenylalanyl-tRNA at 13 mM

Mg++. Under these conditions, [W]phenylalanyl-tRNA or

N-acetyl[W]phenylalanyl-tRNA and [r4C]phenylala.nyl-tRNA

were at the acceptor and the donor site, respectively Using

these complexes, the formation of puromycin derivat ive was

examined. It is clear from this table that the amount of puro-

mycin der ivative of [14C]phenylalanine formed in the absence of

the G factor was greatly reduced when [W]phenylalanyl-tRNA

or N-acetyl[r2C]phenylalanyl-tRNA were added to the reaction

mixture for the additional binding of aminoacyl-tRNA at the

acceptor site. Since the [Wlphenylalanyl-tRNA was bound al-

most exclusively at the donor site, G factor did not appreciably

stimulate the puromycin reaction of this [i4C]phenylalanyl-tRNA

when no additional aminoacyl-tRNA was bound at the acceptor

site. On the other hand, in the presence o f [12C]phenylalanyl-

tRNA at the acceptor site, [W]phenylalanyl-[r2C]phenylalanyl-

tRNA was formed and located at the acceptor site. Conse-

quently, the puromycin reaction of this diphenylalanyl-tRNA

was dependent on G facto r. When N-acetyl[W]phenylalanyl-

tRNA was bound at the acceptor site, no diphenylalanyl-tRNA

could be formed and, consequently, G factor had no appreciable

stimulation on the formation of puromycin derivat ive of [‘“Cl-

phenylalanine. Thus, the inhibitory ef fec t of N-acetyl[W]-

phenylalanyl-tRNA at the acceptor site could be attributed solely

to the steric hindrance.

The data presented in Fig. 2 shows similar eff ects of the [‘“Cl-

phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA present at

the acceptor site on the puromycin reaction of N-acetyl[r4C]-

phenylalanyl-tRNA bound at the donor site.

Since the peptide

bond formation takes place much more rapidly with N-acetyl -

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Issue of January 10, 1972 S. Tanaka, K. Igarashi, and A. Kaji 49

TABLE

III

Effect 01 acceptor site

aminoacyl-tRNA

on reaction

of

donor site

[14C]phenylalanyl-tRNA with

puromycin

For the preparation of the ribosomal complex having [WI-

phenylalanyl-tRNA mostl y at the donor site, the reaction mixture

(1 ml) contained 50 mM Tris-HCl (pH 7.2), 5 mM magnesium ace-

tate, 28 mM NH&l, 12 mm KCI, 200pg of poly(U), 10.8 mg of ribo-

somes, and 9.7 X lo5 cpm (4.95 mg of tRNA) of [W]phenylalanyl-

tRNA. The mixture was incubated and the complex isolated as

described in the text.

The reaction mixture (0.7 ml) for the addi-

tional binding of aminoacyl-tRNA at 13 ml Mg++ contained 20

mM Tris-HCl (pH 7.2), 13 mM magnesium acetate, 40 mM NH&l

and, where indicated, 3 mg of mixture of tRNA containing 1.5

m@moles of [W]phenylalanyl-tRNA or 2.3 mg of tRNA mixture

containing 1.2 mpmoles of N-acetyl[W]phenylalanyl-tRNA.

The mixture was incubated for 20 min at 22”. For the puromycin

reaction of the complex thus prepared, 0.2 ml of the above mixture

was incubated for 60 min at 26’ in 0.36 ml of a solution containing

40 mM Tris-HCl (pH 7.2), 170 mM NH&l; 13 mM magnesium ace-

tate, 1 mM puromycin, 0.2 mM GTP, and where indicated 13 rg of

G facto r. The puromycin derivat ive formed was counted as de-

scribed in the text.

Aminoacyl-tRXA for additional

binding at 13 nm Mg++

Bound

[‘C-

phenyl-

alanyl-

tRNA

[‘~ClPhenylalanyl

puromycin deriv-

atives formed

No aminoacyl-tRNA.. . 736

[W]Phenylalanyl-tRNA..

644

N-Acetyl[W]phenylalanyl-tRNA.

634

Without

G factor G?%r

cm

483 541

107 468

111 136

phenylalanyl-tRNA than with phenylalanyl-tRNA, the rate of

formation of puromycin derivative was studied after a short

incubation period. As can be seen from the figure, at the initial

period of the reaction a marked inhibition of the puromycin

reaction of N-acetyl[“C]phenylalanyl-tRNA at the donor site was

observed. It should be pointed out that no G factor was added

in this experiment. Thus, the inhibitory eff ect of [r’%]phenyl-

alanyl-tRNA at the acceptor site was stronger than that of N-

acetyl[12C]phenylalanyl-tRNA because the former inhibited the

puromycin reaction for two possible reasons, i.e. steric hindrance,

as well as formation of N-acetyldiphenylalanyl-tRNA, while the

latter inhibited only because of the possible steric hindrance.

These results are consistent with the concept that puromycin

attacks the donor site from the direction of the acceptor site.

The experiments described above were based on the assump-

tion that the binding sites for N-acetylphenylalanyl-tRNA were

the same as those for phenylalanyl-tRNA, and N-acetylphenyl-

alanyl-tRNA behaved similarly to phenylalanyl-tRNA with

respect to the binding to ribosomes. To ascertain that this

assumption was correct, the binding sites for N-acetylphenyl-

alanyl-tRNA were compared with those for phenylalanyl-tRNA.

As shown in Table IV, the amount of bound N-acetyl[W]phenyl-

alanyl-tRNA was approximately equal to that of [r4C]phenyl-

alanyl-tRNA at three different Mg++ concentrations tested.

The addition of an equal amount of [lZC]phenylalanyl-tRNA

reduced the binding of [14C]phenylalanyl-tRNA as well as N-

acetyl[14C]phenylalanyl-tRNA. It is noted that in the presence

presence of high Mg++, [i2C]phenylalanyl-tRNA inhibited more

effi cien tly the binding of N-acetyl[14C]phenylalanyl-tRNA than

that of [14C]phenylalanyl-tRNA. This suggests that the binding

TABLE

IV

Evidence that binding sites for N-acetylphenylalanyl-tRNA are same

as

those

for phenylalanyl-tRNA

The reaction mixture (0.1 ml) for the binding of [14C]aminoacyl-

tRNA contained the following in micromoles: 8.0 Tris-HCl (pH

7.1), 4.0 KCl, various concentrations of magnesium acetate, 30

rg of poly(U), 160 rg of ribosomes, and 16,800 cpm (38.2 rpmoles) of

[r4C]phenylalanyl-tRNA or 16,800 cpm (38.2ppmoles) of N-acetyl -

[W]phenylalanyl -tRNA. In some cases, a mixture of tRNA con-

taining 38.2 ppmoles of [lzC]phenylalanyl-tRNAor 38.2 ppmoles of

N-acetyl[W]phenylalanyl-tRNA was added. The reaction was

incubated for 25 min at 22” and the bound radioactive aminoacyl-

tRNA was assayed by the Millipore filter technique.

Aminoacyl-tRNA used

N-acetyl[W]phenylalanyl- tRNA

[14C]Phenylalanyl-tRNA.

N-Acetyl[14C]phenylalanyl-tRNA and

[12C]phenylalanyl-tRNA. .

[W]Phenylalanyl-tRNA and [‘WI-

phenylalanyl-tRNA

[r4C]Phenylalanyl-tRNA and N-ace-

tyl[W]phenylalanyl-tRNA.

Bound aminoacyl-tRNA

5rnM 13 nlY

I 1

21 InM

MC+ Mg*

Mg++

1102

1152

@m

4869

5802

498 2177

503 3913

1138 6043

6GG4

6612

2381

4290

6532

affi nity of phenylalanyl-tRNA is higher than that of N-acetyl-

phenylalanyl-tRNA under these conditions. When both [‘“Cl-

phenylalanyl-tRNA and N-acetyl[14C]phenylalanyl-tRNA were

present in the reaction mixture, the total amount of bound

radioactivity was approximately the same as that with [‘“Cl-

phenylalanyl-tRNA or N-acetyl[14C]phenylalanyl-tRNA alone.

These observations show that the binding site for N-acetyl -

phenylalanyl-tRNA is propably identical with that for phenyl-

alanyl-tRNA at low (5 to 6 mM) as well as high (13 mrvr) Mg+f

concentrations. Thus, one can conclude that in the presence of

low Mg+f, N-acetylphenylalanyl-tRNA binds mostly to the

donor site while it ‘binds to both donor and acceptor sites at

high Mg+f.

DISCUSSION

Preceding reports on the action of protein synthesis inhibitor

(9) were based on the assumption that in the presence of low

Mg++, binding of phenylalanyl-tRNA to ribosomes took place

mostly to the donor site while both donor and acceptor sites were

occupied with phenylalanyl-tRNA in the presence of high Mg+f.

These assumptions were derived from the results of the NH2-

terminal analysis of the polyphenylalanine formed from the

complex of [r4C]phenylalanyl -tRNA, poly(U), and ribosomes at

different concentrations of Mg++ (2). Although a similar con-

clusion was obtained from the studies on the puromycin reaction

of the ribosome-bound phenylalanyl-tRNA (7), it was necessary to

obtain fur ther evidence for this notion by identifying the product

of puromycin reaction.

In this discussion, we designate the complex made in the pres-

ence of low Mg++ (5 to 6 mM) as complex A and that made in

the presence o f high Mg++ (13

nlM)

as complex B. The analysis

of the puromycin reaction product clearly established that the

bound phenylalanyl-tRNA of complex A was mostly located at

the donor site because the addition of G factor resulted in very

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50

Action of Tetracycline and Puromycin

Vol. 247, No. 1

small, if any, stimulation of the formation of diphenylalanyl

puromycin.

On the other hand, complex B yielded diphenyl-

alanyl puromycin as well as phenylalanyl puromycin in the

presence o f G facto r.

These results give further support of the

previous notion (2) that in the presence of low MgH (condition

A) only the donor sites are occupied with phenylalanyl-tRNA,

whereas both acceptor and donor sites ore occupied with phenyl-

alanyl-tRNA in the presence of high Mg+f (condition B).

With the analysis of the puromycin derivatives formed from

the bound phenylalanyl-tRNA, it became clear that, in confuma-

tion of our previous results with NHz-terminal analysis o f

polyphenylalanine (7), tetracycline inhibited significantly the

bindmg of phenylalanyl-tRNA at the donor site as well as the

acceptor site in the presence of high Mg* (condition B). In the

presence of

low

Mg* and T factor the action of tetracycline was

relatively speci fic to the acceptor site. It should be pointed out,

however, that in the normal situation, the donor site is occupied

with either unesterified tRNA or peptidyl-tRNA; thus the

possiblity that the aminoacyl-tRNA directly binds to the donor

site does not exist in the normal protein synthesis.

Only when

the empty ribosomes are presented to an aminoacyl-tRNA with

synthetic messenger RNA, such a situation takes place.

The presence of N-acetylphenylalanyl-tRNA or phenylalanyl-

tRNA at the acceptor site has a strong inhibitory ef fec t on the

puromycin reaction o f the donor site-bound [14C]phenylalanyl-

tRNA. The inhibition by the presence of [Wlphenylalanyl-

tRNA at the acceptor site is due to two reasons.

The first is

formation of [14C]phenylalanyl-[W]phenylalanyl-tRNA bound

to the acceptor site. In support of this, a marked increase of

the puromycin reaction was observed by the addition of G factor.

The second reason for the inhibition is a possible steric hindrance

caused by the presence of aminoacyl-tRNA at the acceptor site.

This notion was supported by the observation that the presence

of N-acetyl[W]phenylalanyl-tRNA at the acceptor site had

strong inhibitory eff ect on the puromycin reaction of the donor

site-bound [14C]phenylalanyl-tRNA. The inhibitory eff ect of the

acceptor site-bound N-acetyl[12C]phenylalanyl-tRNA could be

due to its effect on the conformation of the ribosome.

At any

rate, these observations indicate that puromycin attacks the

donor site peptidyl-tRNA from the side of the acceptor site.

Al-

though there is no direct evidence that puromycin is actually at

the acceptor site when it reacts with the donor site peptidyl-

tRNA, our present observation is consistent with the current

notion that puromycin will react with peptidyl transferase in a

way similar to the reaction of acceptor site aminoacyl-tRNA.

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action of tetracycline and puromycin

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