Vol. 65, No. 2, 1975

ON

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE

COVALENT

PART

OF

THE

mRNA

BINDING

BINDING

16

S RNA

SITE

Rolf

THE

and

Hans

Technischen

D

61

IS 30

Fachgebiet der

mRNA

WHICH

OF

Wagner

OF

MODELS

LOCATED

TO IN

THE THE

S RIBOSOME

G~nter

Gassen

Biochemie

Hochschule

Darmstadt,

Darmstadt

PetersenstraBe

15

Received May ].5,].975 SUMMARY !

The oligonucleotide U - U - i 3' H [ U'- ( n h 2 ) 2 U as a m R N A model was bound covalently to t h e 16 S R N A v i a the bifunctional guanosine-specific reagent pchlorocarboxyphenylglyoxal. Since the ribosomes labeled with the oligonucleotide stimulated the binding of Phe-tRNA, it is c o n c l u d e d , that the probe reacted specifically with a guanosine of t h e 16 S R N A w h i c h forms the mRNA binding s i t e of t h e 30 S r i b o s o m e . The binding of t h e p r o b e w a s inhibited by poly(U) , but stimulated by the pres e n c e of P h e - t R N A . 83 % of t h e r a d i o a c t i v i t y was f o u n d i n t h e 16 S R N A of t h e r i b o s o m a l subunit. After digesting t h e 16 S R N A w i t h r i b o n u c l e a s e T 1 and separating the oligonucleotides formed by gel electrophoresis, the radioactive labeled material was mainly f o u n d in o n e d i s t i n c t band.

INTRODUCTION The

topography

spatial 16

S RNA

is

research by

at

the

each Much

less

role

of

and is

the

of to

known 16

S ribosome,

the

a

methods

the

field i.e.

about

of

the

and

the

intensive

crosslinking labeling,

RNA

and

protein

on

the

identification

proteins 16

namely

proteins

affinity

center the

S RNA.

Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.

19

present

between

methods

location

other

30

of

reagents,

formation

munological the

the

] I-3 i . M o s t

bifunctional

complex

of

of

arrangement

with

and

respect

im-

to

S RNA. the

Most

519

possible

chemical

catalytic modification

Vol. 65, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

experiments

result

tion

ribosome

ted

of to

the

a direct

change

in

complex is, 16

however,

le

of

in

the

nition ry

the

binding

very

little

which

we

to

covalently the

the

to

ribosome.

16

of

covalent

binding

template

activity

the

an

and

in

the

Although and

the

of

we

have

part

of

191.

in

of

30

the

pronow

the

16

S RNA

order 16

to

S RNA,

mRNA

models

aminoacyL-site

following

the

prima-

its to

In

as

ro-

up

the

the

the

recog-

the

many

of

of

important

we

report

oligonucleotide

to

There

aminoacyl-

regions

the

the

part

S RNA

In

to

altered

protein.

oligonucleotides

the

or an

the

aminoacyl-site

bind

synthesis

RNA

the

known,

exposed

to

the due

attribu-

that

mRNA

of

be

func-

within

S RNA

are

certain

and

14-81.

16

knowledge

these

of

the

the

of

plays

the

AA-tRNA

identify tried

of

a

either

RNA

located

sites

forms

may

S ribosome

of

tein

of

evidence,

binding

of

and

between

is

30

sequence

loss

conformation

good

which the

a

involvement

protein

formation

S RNA

site

in

on

probe,

S ribosome

the

and

oligonucleotide

the labeled

ribosomes. MATERIALS

AND

METHODS

13HIUridine-diphosphate were

obtained

Amersham from MRE

U.K.,

600

by

standard thr~gh 0.5

M

ted

in mM

35

i0 KCI

fugation vating 20

mM

Merck

%

mM

S

Tris-HCl

and

0.2

formed

mM

were

I ii I . T h e buffer

and

70

(20

Tris-borate

(EC

were

7.2,

the

separated

subunits

were

mM

Mg(OAc)

2,

pH

7.2) a t

-80°C.

520

once

30

with

M g C I 2, S

and

zonal stored

200

by

dissocia-

mM

by

The

cells

were 0.7

were

sedimented

washed

EDTA-K;

cells

coli

ribosomes pH

coli

Darmstadt. E.

were

2.7.7.26)

luteus

Company, f~om

Centre,

Escherichia

M.

! IO I . T h e y

sucrose The

T 1 and

and

isolated

procedures

NH4CI.

subunits

phase)

the

were

13HIphenylalanine

Radiochemical

Mannheim,

(mid-log

supplied

and

the

ribonuclease

Boehringer,

ribosomes

20

from

mM

50

in

NH4CI

Prior

S

centri-

to

actiand use,

Vol. 65, N o . 2, 1 9 7 5

they me

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

were

activated

for

[12 I . T h e

synthesis

buffer

25 m i n

deoxyuridine-5'-diphosphate azido-2'-deoxyuridine procedure

published

reduction

of

the

L~hrmann

act.

with

polynucleotide

aid

of

The

nylchlorid

to The

phosphate the

were

for

gelfiltration the

probe

on

is

et

the

al

1141

with

the

last

step

synthesized

reacted

with

-

1151 . thio-

p-chlorocarboxyphenyl-

7.8

dissolved

a n d an e t h e r vigorously

Excess

G-25.

in F i g .

in

0.5

solution at

reagent

Sephadex

depicted

was

phosphorylase

was

shaken

iO m i n .

2'-amino-2'2' U) f r o m 2 ' closely

as

sa-

20 C i / m o l e )

the

pH

the

U-U-13HIU

oligonucleotide

buffer

reagent

rature

yield

in

oligonucleotide

p-Carboxylicphenylglyoxal

glyoxal.

the

followed

the reaction. 2' (nh 2) U (spec. the

of

2'-azidogroup

of

40°C

( p p ( n h 2)

I131 by

at

room

was The

M of

tempe-

removed

by

synthesis

of

I.

RESULTS In

order

was

to

test

incubated

spectively

the

with

(Fig.

reactivity

poly(U,G)

2) . A b o u t

of

or 600

the

with pmoles

probe,

it

G-G(-U) 7 reof

U-U-!3HIU

CH3_I_O_CN KOH ~ I~ SeOz, HC-C / ~ ' (~ o . ,,- ('(" ~ ")/~-c-0H )CH3-~ 0II 0/ -~--~-c-°H SOCti"k)

d~H

HO

=:(u-u-[,HIu-)

" ~

H2N Rb o

HO~

HO NH2

HO-,

~°~2"\/

ROy ~ r0

_

\

/

/

"~-i"

U I

.o oo

Fig.

1 CHEMICAL BINDING

SYNTHESIS OF THE PROBE AND ITS THE GUANINE BASE OF A RNA.

TO

521

-

Vol. 65, No. 2, 1975

BIOCHEMICAl AND BIOPHYSICAL RESEARCH COMMUNICATIONS

~0o-

x

: G - G (-U)7 ~ p01y (U,G)

,

,

:

c-

g

..Q "D

~ soo LON

i

D

,

,

,

,

,

50

100

U-U~aH] U-(nhr) ZU [n moles] Fig.

2 BINDING OF CONTAINING

U-U3HIU-(nh)2' OLIGONUCLEOTIDES

The reaction mixture pH 7 . 2 ) contained in

U TO G U A N O S I N E .

( 2 0 mM T r i s - b o r a t e a total volume of

30 ~/i 0 . 2 A 2 6 0 - U . poly(U,G) or 0.2 A260-U. G-G(-U) 7 and was incubated for 1 h at 37°C. The polynucleotides were separated from excess reagent by paper chromatography in 1 M NH4OAc : C2H5OH 1 : i .

2 1

(nhr)

U

were

efficient

bound

binding

stabile

to to

2 nmoles

G-G(-U)

poly(U,G)

may

of

be

secondary

structure

this

the

common

nucleosides

The

tetranucleoside

7. due

The to

less the

polynucleotide

1161 None

of

measurable with

a

other extend.

terminal

activity

which

U-U-U-U,

but

may

be

group.

For

absence sence of

is

by

of

reagent

the

covalent with

of

about

comparable

caused

incubated

2'-aminouridine

30

Phe-tRNA

saturating were

50

%

U-U-U

bound

lower

pK-value

the

as

shown

amounts to

500

522

compared

in

the Fig.

of

to

3) . T h i s the

2'-amino-

presence 4.

Phe-tRNA

pmoles

a

template

oligonucleotide

in

of

a

(Fig.

(5.2)

ribosomes

to

triphosphate

shows

to

binding S

reacted

of

In

the

250 30

S

was and pre-

pmoles ribo-

Vol. 65, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1000P3

o

o U-U-U

:

-- U - U - U - U

o

\

Z

~

25-

112 m

.

,

,

",'

.

.

,

.

,

~

100

oligonucleotides [ nmoles ] Fig.

3 STIMULATION OF 13H]Phe-tRNA BINDING RIBOSOMES IN P R E S E N C E OF 3'-TERMINAL URIDINE-CONTAINING OLIGONUCLEOTIDES.

T O 30 S AMINO-

The following mixture (total volume iOO ~i) was incubated f o r i h at O ° C : i0 ~ i 30 S ribosomes (80 A 2 6 0 - U . / m l ) , 30 ~ i ! 3 H I P h e t R N A (40 A 2 6 0 - U . / m l , spec. activity iOO0 Ci/Mol, charged 1.6 %), 50 ~ l m i x (20 m M M g ( O A c ) 2, 1 5 0 m M N H 4 C I , 50 m M T r i s b o r a t e p H 7.2) a n d t h e a m o u n t s of o l i g o nucleotide indicated. Otherwise the proc e d u r e of N i r e n b e r g and Leder was foll o w e d 1181.

somes 250

within

should

be

codon-tRNA der

to

the

mRNA

ing As

bound

that

binding the

O°C.

About

40

noncovalently formation

the

pmoles by

(see

site

of

the

30

S,

3) . In

was it was

oligonucleotide-labeled

can

be

seen was

U-U-U-(nh

2)

for

2'

the

in

Fig.

bound U

by

after times

5 three

preincubating indicated.

523

With

to

tested

ribosomes

times 2' U

U-U-U-(nhr)

or-

bound

Phe-tRNA. Stimulation of P h e - t R N A 2' U - U - U - ( n h 2) U w a s u s e d as a c o n t r o l .

Phe-tRNA

of

ribosome-

Fig.

oligonucleotide

out

bind

by

somes

at

complex

show

whether could

1 h

the

amount

compared

the

30

bind-

of

to

S ribo-

U-U-U-(nh

2)

2'

U

Vol. 65, No. 2, 1975

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

.~ in presence of Phe -tRNA x in obsence of Phe-tRNA (3.

!

no c.(D

200-

no o 0 100-

c 0

~g

10

~

100 U - U - [ 3 H ] U - ( n h r ) ZU [nmoles]

Fig.

4

COVALENT SOMES IN tRNA.

BINDING OF THE PROBE TO THE PRESENCE AND ABSENCE

30 S RIBOOF Phe-

The reaction mixture was incubated for 1 h at OOC to minimize nuclease degradation of the oligonucleotide. With the exception that [3HIPhe-tRNA was replaced by Phe-tRNA the same conditions were used as d i s c r i b e d under Fig. 3.

as

template

30

min,

continued As

a plateau

wherease to

further

bound

incubated

The

ribosomes

and

were

-5°C. reagent

was

manner.

fer,

Phe-tRNA

ribosomes

the

1 h in

at

was

(see After was

at

activity

O°C.

shown

30 for

precipitated 30

OOOxg

washed

with

in

table

i)

and

The in

table

524

i.

O°C. ethanol

%

20

min

%

at

ethanol

supernatant.

the

template

at

65

65

the

ribosomes

1 h

for

were

the

S

of

with

the

suspending added

after 2' U

4 h.

codon

reagent

reached

U-U-U-(nhr)

after

the

undetectable

same

was

of

oligonucleotide with

pellet

ribosomes

for

for

were

trol

ted

even

centrifuged

The

binding

activity

increase

evidence

covalently were

of

the

treated

Conin

activity

the in

ribosomes

mixture

until

was of

buf-

incubathese

Vol. 65, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

50.

x--x U - U- U-inhr} 2'U

,--.

U- U-U-Inh2} 2"U

25-

-1-

r~

6b

Fig.

5 STIMULATION OF VALENTLY BOUND

16

S RNA

and

show

were

of

ted

the

aqueous

in

phase

was

a

the

tRNA.

to

the

30

hydrolysed

to

with

the

ribosomal

ribosomes at

O°C

the with

S

CO-

suspended citrate-Na, 1 %

SDS)

over

a

same

of

12

ribosomes,

protein.

The

T 1 and

525

nmoles 83

%

~i

mM

EDTA-K, extrac-

buffer.

the

The

G-IOO was

co-

used

probe

was

4 nmoles to

labeled the

500

were

tRNA of

of

and

iO

Sephadex

proportion

pro-

nm01es

in

and

with

for

Following

centrifugation

unfractionated

Out

300

2 h.

mM

passed

RNase

and

for

were

7.2,

large

to

%

specific

equilibrated

bound 17

S

15

6) . S i n c e

assay,

was

ethanol,

pH

bound

and

30

NaCI,

phenol

(Fig.

the

BY

probe

react

ribosomes mM

Tris-borate

with

lumn

with

(150

mM

not

incubated

precipitation

buffer

that

did

3 nmoles

washing,

13HIPhe-tRNA BINDING U-U-U-(nhr) 2'U.

the

to

reagent

2~ t [min]

mixture w a s t h e s a m e as d i s F i g . 3. T h e t o t a l v o l u m e , increased to i m l . i O O ~ i withdrawn at the time interand were assayed for binding.

order

teins

~

The reaction cribed under however, was aliquots were vals indicated [3H[Phe-tRNA

In

50

i~

were

the

16

S RNA

16

S RNA

was

oligonucleotides

Vol. 65, No. 2, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

cpm

A260-U

1500

--A2so-U . . . . .

cpm 1000

i

....../ I0

20

3o

~'o

fraction number Fig.

6

SEPARATION OF LABELED SEPHADEX G-IOO COLUMN.

16

S RNA

ON

A

The aqueous phase obtained from phenol extraction of the labeled 30 S ribosome was passed over a Sephadex G-IOO column (i c m x 50 cm) . I t w a s e l u t e d with 50 mM Trisborate pH 7.2 in the cold. Aliquots were assayed for radioactivity on glasfiber filters.

TABLE

i

J3HIPhe-tRNA RIBOSOMES.

BINDING

TO

OLIGONUCLEOTIDE

LABELED

30 S ribosomes were incubated at O°C for 1 h in the presence of e x c e s s reagent. 50 pmoles o f 30 S were removed to m e a s u r e 13HIPhe-tRNA binding. To control 3 saturating amounts of p o l y ( U ) were add e d to t h e 30 S r i b o s o m e s to t e s t f o r r e m a i n i n g activity.

labeled 3OS probe p o l y (U)

I3HI P h e - t R N A

1 control 3OS

2 control 3OS

-

U-U-U- (nh 2) 2 'U

U-U-U- (nhr) 2 'U

3 control 3OS

-

-

-

+

+

+

+

+

5.4 pmoles

O. 2

O. 4

15. O

13 IP e-t A bound/ 5Opmoles 30 S

526

Vol. 65, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cprr

~0

II,

I I

Fig.

I

I 2

II 3/.

I 5

II 67

• • 8 g

I 10

I ~)

7 SEPARATION OF O L I G O N U C L E O T I D E S OBTAINED FROM RNase T1 D I G E S T I O N OF L A B E L E D 16 S R N A O N A 12 % P O L Y A C R Y L A M I D E GEL. The electrophoresis was performed a t 4 0 0 V, 25 m A / t u b e f o r 3 h. F o l l o w i n g staining in methylene-blue and destaining f o r 12 h, the bands were sliced and counted following known procedures.

obtained gel

were

1171 . F i g .

dioactivity. is

separated

found

7

on

shows

The

main

in

band

to

identify

a

the

12

% polyacrylamide

distribution

portion

of

the

of

the

ra-

radioactivity

5.

DISCUSSION In

order

16

S RNA

site

of

glyoxal with

which the

are

30

S

modification

in

making

possible. consists

of

an

in

the

of

the

that

the

ribosome.

the

selected

mRNA

reacts

guanine

of

binding

119,201.

the

In

of to

a

second

527

mRNA step

acid

T 1 resistant,

oligonucleotide

of

the

reagent it

Phe-tRNA

the

It

guanylic

uridine-oligonucleotide presence

the

a phenyl-

RNase

part

of

specifically

of

becomes

second

electrostatically

the

in

advantage it

areas

we it

identification Since

be

bound

since

hydrogen-bonded shows

step

located

derivative,

non

stranded

ribosome,

furthermore

thus

single

in

binding the

should

the

first site

glyoxylic

Vol. 65, No. 2, 1975

part

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

moiety Since

the of

the

the

ing

the

ent

with

tein

S

16

S RNA

ed

by

with

the

guanine

our

forms

that

data

also

analogue

gel

the of

bound binding

the

mRNA

bind-

electrophoresis

should

121,221.

a complex

and

a mRNA

the

labeled

previous

]2[,

actively

neighborhood to

S RNA

still

concluded

According

15,

16

be

close

oligonucleotide the

react

RNA.

it may in

site.

C of

should

oligonucleotide

Phe-tRNA, occured

reagent

be

This which with

becomes

located

would

be

showed part

in p a r t consist-

that

C of

covalently

affinity

label

the

pro-

the label-

123 I .

ACKNOWLEDGEMENT This work was supported schungsgemeinschaft and schen Industrie.

by the Deutsche Forthe Fonds der Chemi-

REFERENCES i.

P o n g s , O. , N i e r h a u s , K.H. , E r d m a n n , V.A. , and Wittmann, H.G. (1974) F E B S L e t t . 40, 2 8 - 3 7

2.

Ungewickell, E., G a r e t t , R., E h r e s m a n n , C., Stiegler, P., a n d F e l l n e r , P. (1975) E u r . J. Biochem. 51, 1 6 5 - 1 8 0

3.

Morgan, J. a n d B r i m a c o m b e , Biochem. 37, 4 7 2 - 4 8 0

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Zimmermann, R . A . , M a c k i e , G . A . , M u t o , A. , Garrett, R.A., Ungewickell, E., E h r e s m a n n , Stiegler, P., E b e l , J . - P . , a n d F e l l n e r , P. (1975) N u c l e i c A c i d s R e s . 2, 2 7 9 - 3 0 2

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(1974)

R.

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Biochemistry

Gette, W.R., and 13, 2 1 1 5 - 2 1 2 2

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Held, W.A., Biochemistry

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Schwartz, I. a n d N a t l . A c a d . Sci.

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S h i n e , J. a n d D a l g a r n o , L. (1974) A c a d . S c i . U . S . 71, 1 3 4 2 - 1 3 4 6

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Budker, V.G., Girshovich, A.S., Grineva, N.J., Karpova, G.G., Knorre, D.G., and Kobets, N.J. (1973) D o k l . A k a d . N a u k . S S S R 211, 7 2 5 - 7 2 8

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G r o s , F. Molecular

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L e i f e r , W. a n d K r e u z e r , Biochem. 44, 8 9 - 9 6

T.

(1971)

Analytical

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Z a m i r , A. , M i s k i n , R., a n d J. M o l . B i o l . 60, 3 4 7 - 3 6 8

Elson,

D.

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Verheyden, (1970) J.

J.P., Wagner, Org. Chem. 36,

528

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(1971)

Moffatt,

J.G.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14.

L~hrmann, R. , S c h w a r z , U., a n d (1973) F E B S L e t t . 32, 5 5 - 5 8

15.

Schetters, H., G a s s e n , H.G., and Matthaei, (1972) B i o c h e m . Biophys. A c t a 272, 5 4 9 - 5 5 3

16.

Thang, M.N. and Grunberg-Manago, M. (1968) Methods in E n z y m o l o g y Vol. X I I A c a d e m i c Press 512-529

17.

Wachter, M . W . a n d F i e r s , W. Enzymology Vol. XXI Academic

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Nirenberg, M.W. 145, 1 3 9 9 - 1 4 0 7

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