Stimulation of the binding of aminoacyl-sRNA to ribosomes by tubercidin (7-deazaadenosine) and N6-dimethyladenosine containing trinucleoside diphosphate analogs

Vol. 21, No. 3,1965

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

STIMULATIONOF THEBINDING OF AMINOACYGsRNATO RIBOSOMES BY TUBERCIDIN(7-DEAZAADFNOSINE)ANDN6-DIMECHYLAD~OS~E CONTAININGTRINUCLEOSlDE DIPHOSPHATE ANAZOGS Morio Ikehara and Eiko Ohtsuka Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan and Institute for Enzyme Research, University of Wisconsin, Madison

Received

October

4, 1965

It has recently

been shown that ribotrinucleotides

for the binding of specific 1964; Nirenberg & &.,

serve as templates

aminoacyl-sRNA's to ribosomes (Nirenberg et al.,

1965; 311 et al.,

1965).

This specificity

of binding

is presumedto be brought about by hydrogen bond formation between the bases of the trinucleotide therefore

and the anticodon bases of the sRNA. It was of interest

to investigate

the stimulation

directed by trinucleotides

of aminoacyl-sRNA binding to ribosomes

containing base analogs in which this hydrogen

bond:ng may be affected.

For this purpose we have taken the trinucleoside

di-

phos:)hates ApApA* and ApCpC, which serve as codons for lys-sRNA and thr-sRNA resp:ctively, and replaced the 5'-adenosine end of each chain with N6.-dlmethyladenlsine (Ikehara et al., et al.,

1960; Y. Mizuno et al.,

to be involved al.,

1961) and tubercidin 1963).

(7-deazaadenosine)(S. Suzuki,

As the 6-amino group of adenine is known

in hydrogen bond formation with the 4-O atom of uracil

(Rich &

1956), conversion of this group to the dimethylamino group should eliminate

this bonding.

The fact-that

double stranded helical substantiates this idea. only in the substitution

polar

N6-dimethyladenylic

complex with polyuridvlic

acid fails

acid (Griffin

Tubercidin on the other hand differs

to form a et al.,

1964)

from adenosine

of the N7 of the adenine by CH. Testing the stimulation

* Ak:breviations: ApApA, adenylyl-(3~+5~)-adenylyl-(3~;f5')-adenosine; A, aderiosine; G, guanosine; C, cytidine; U, uridine; DMA, N -dimethyladenosine; Tu, tubercidin. 257

Vol. 21, No. 3, 1965

BIOCHEMICAL

KN-H

AND BlOPHYSlCAL

tubercidin

(Tu)

action

of lys-sRNA

in the were

These

possible

that

using

with

Therefore those

DMApCpC were

compared

The other

trinucleoside

chemically

(Lohrmann

sRNA's

A partially

current

distribution

glu-sRNA. &

with

purified

Cl'-pro,

of C14-amino 165;

with acids

C14-lys,

used were: 222;

C14-ser,

into

with

RNA

1964).

G, C or U rather

It

than

A

DMApApA and TupApA

Similarly

using

the

binding

studies

GpC@.?, CpCpC and UpCpC.

TupApA,

(Ikehara in this

of sRNA which

a given

in the

the mode of

(Acs et al.,

TupCpC,

et al., study

has been described

et al.,

a role

clarifying

1965) were amino Cl'-ala,

also

of all

synthesized C14-aminoacyl-

previously

(53811 et el.,

was obtained

by counter-

was used for prepared

the preparation

by charging Specific

acid. 123;

123; C14-thr,

258

DMApApA and

in preparation).

were

The preparation

aminoacyl-sRNA's

B sF?NA preparation

plays

and

AND METHODS

used

fraction

(Nishimura

those

TupApA

to be incorporated

for

analogs,

in press).

B ribosomes

The remaining

(~c/~mole) 205;

et al.,

known

studies

synthesis

diphosphates

and of g. &

1965).

&

by chemical

triplets

in

CpApA end UpApA.

diphosphate

obtained

is

substitute

MATERIALS The ribotrinucleoside

of interest

the binding

of GpApA,

DMApCpC and TupCpC were

by the

in mouse fibroblasts

DMA or Tu could

trinucleotide. compared

are which

synthesis

adenosine

the N7 of adenine

also

tubercidin

protein

(DMA)

to ribosomes

show whether studies

antibiotic

and to inhibit was also

and thr-sF!.NA

should

bonding. of the

OH

N6-dimethyladenosine

TupCpC respectively hydrogen

H 'N' H

H3GN.CH3

HO

of binding

RESEARCH COMMUNICATIONS

C14-gin, 164..

unfractiona

activities 32.2;

of

Cl'-glu,

For the binding

.ted

Vol. 21, No. 3, 1965

BIOCHEMICAL

of sRNA to ribosomes, incubation unless

the method

mixture

(0.05

otherwise

where

used

ire

shown for

out a; 25'

for

0.1 M buffer,

routinely

specified.

unless

(1964) was used.

units

0.05

The concentrations in the

otherwise

M KCl,

of ribosomes

used was sodium

each aminoacyl--sRNA

20 min.

RESEARCH COMMUNICATIONS

and Leder

1.5 - 2.0 O.D.

The buffer otherwise

of Nirenberg

contained

indicated,

of trsnucleotides. excep-;

ml.)

AND BlOPHYSlCAL

0.02

M Mg(OAc)2

and Y-12 mmoles

cacodylate-HCI,

of labeled

Table.

The

pH 7.2

aminoacyl-sRNA's

The incubations

were

carried

specified.

RJEULTS AND DISCUSSION &ile

the trinucleoside

sRNA to ribosomes, for

template

the

diphosphate

analog

activity

DMA cannot

replace

either

hydrcgen

of codon

to anticodon,

with

binding.

groq

interferes

Other

experiments

binding

(Table

in tlie

on the

codons

binding

tested.

-;rinucleotide

In the

some3 of C14-thr-sRNA, zodons

activity

are

unable

ribosomes,

while

(Table

showing

codon.

of sla-sRNA, onl>'

for

this

to test

triplet

fails

same way DMApCpC failed

to replace

analog

the bulk

of the

not

stimulating

that

tubercidin

TupCpC stimulated ser-sRNA

the

binding

respectively,

whether

or pro-sRNA,

the

again

showing

A.

259

that

G or C U in

binding

of any

the binding for

Thus for

to ribowhich

template

trinucleotides.

binding

for

the

DMApApA had neither

to stimulate

of C14-lys-sRNA

A but

of C14-thr-sRNA

the binding

preferred.

DMA can replace

of C14-gin-sRNA

can substitute

is

GpApA and CpApA stimulated

to stimulate

the

result

dimethylamino

C14-ser - sRNA and Cl4 -pro-sRNA,

stimulated

that

in the

the former

A, G, C or U in these

TupApA

This

Thus DMA can replace

not possible

Cl'-ala-sRNA,

The tubercidin

this

is

showing

of the 6-NH2 group

and C14-gin-sRNA sRNA's.

I)

trinucleotide.

explanations

whereas

of C14-lys-

binding

(Table

ApCpC, GpCpC, UpCpC and CpCpC respectively.

DMA is

I),

Of these

of these It

or that

showed that

UpApA since

aminoacyl-sRNA.

the

I)

A in this

of the importance

C14-glu-sRNA

to ribosomajof

no e!'fect

the

in terms

the

the

DMApApA gave no stimulation

may be explained bonding

ApApA stimulated

or C14-glu not but

tubercidin

for not

to - sRJJA

C or G in the binding

substitutes

UPAPA CPAPA Control

1.77 0.78 0.52

0.48

0.63

0.46

0.49 0.68

DMApCpC GPCPC UPCPC

CPCPC Control

0.07

0.05 0.04

0.15 0.16 0.18

0.16

0.74

0.55 0.14

0.16

0.23

0.15

0.16

0.23

0.18

1.42

1.44

1.76

1.37 1.66

8.32+-E 6.4EW

0.51

0.59

0.49

0.97

0.54 0.56

o-55 0.57 0.86

0.53 0.56

0.53 0.52

C14-Ser sRNA+w+c (22.0 Wmoles) 6 mole 12 mole

0.05

0.06 0.93

0.06

0.04 0.97

0.04 0.13

0.42 0.31

0.29

0.35 0.35 0.35

0.30

0.39 0.36 0.44 0.33 0.40 0.40

C14-Pro-sRNA (4.8 IztYnoles) 6 mole 12 mole

1.87 1.72

2.63

1*35

7.711"'

8.2FJH:-

C14-Lys-sRNA (17.9 Wmoles) 6 vole 12 mole

by Triribonucleotides

Buffer used was sodium cacodylate-HCl, pH 7.2 for 0.02 M M~(OAC)~. Incubations were at 25' for 20 min. Other conditions, see text. $5For accuracy these experiments were carried out on twice the scale, as gln-sRNA had a low specific activity. +e+ Control value was 1.04. SW Since the binding stimulation in cacodylate buffer was too small, tris chloride, pH 7.2 was used.

2.11

1.94 0.55 0.48

Cl4-Thr-sRNA (19.2 pvmoles) 6 wmole 12 mole

1.91

APCPC TupCpC

0.75

WpA

0.08

0.07

C14-Glu-sRNA (2.96 w-moles) 6 mole 12 mole

to Ribosomes Stimulated

Cl'-Ala-sRNA (6.5 trwnoles) 6 wmole 12 mole

1.19

DMApApA

2.92 0.76

0.83 1.31 0.77 1.31

0.79

TupApA

APAPA

0.73 0.77

~144~-s~A” (40.8 uwnoles) 6 mole 12 mole

of Aminoacyl-sRNA

0.77 0.75

Binding

TABIEI

Vol. 21, No. 3, 1965

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

While the extent of the binding of C14 -thr-sRNA stimulated by TupCpCand ApCp: was similar

(Fig. l),

TupApA stimulated C14 -lys-sRNA binding to a smaller

mlApcpc f d 3

0 Fig. 1.

I

I

12 TRIN3UCLEOTIDGES

hph4oLES)

Stimulation of the binding of thr-sRNA (19.2 #moles) to ribosomes by trinucleotides. Conditions are described in Materials and Methods.

-PAPA

Fig. 2.

Stknulation of the binding of lys-sRNA (17.9 moles) to ribosomes by trinucleotides. Conditions are described in Materials and Methods.

261

Vol. 21, No. 3, 1965

extent

than

did

ApApA at 25'

(37') and lower lys-sHNA

than

binding

BIOCHEMICAL

Mg did

tion.

concentration,

of the

of the

type

DMApApA was again

binding

3).

hydrogen

bonding

inactive

at the

2).

TupApA

A possible

At higher

gave better

explanation

for

may be an effect

could

be that

are involved higher

temperature of C14 -

binding the

difference

hydrophobic in this

temperature

DMApCpC, TupCpC and ApCpC all

phenomenon

3).

of the

bond

interactions

(Fig. failed

in

of the replacement

(1953) type hydrogen

by CH on Matson-Crick

explanation

concentration

d+

(Fig.

however,

trinucleotides

adenine

An alternative

Watson-Crick

and 0.02 M Mg*

ApApA (Fig.

capacity

N7 position

-I+

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

forma-

or non

to some extent.

At 37' and 0.01 M

to stimulate

C14-thr-sHNA

to ribosomes.

Fig.

Stimulation of the b&nding of lys-SHEA (17.9 wales) to ribosomes by trinucleotides at 37tF Incubations were for 8 min. in Tris-HCl buffer pH 7.5 and O.OlMMg . Other conditions are described in the text.

3.

Triplets

containing

been shown to serve indicate translation of protein

that

the

as templates

incorporation step

of protein

synthesis

antibiotic

tubercidin

in the binding

of tubercidin synthesis.

by tubercidin

into If

(Acs

this

et al.,

262

at the

St-end

position

of sRNA to ribosomes. mRNA does not is

so then

interfere

the rapid

1964) must occur

have This

may

with

the

inhibition

at another

step

Vol. 2 1, No. 3, 1965

in the

protein

being

BIOCHEMICAL

synthesis.

synthesized

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Tubercidin

order

containing

to demonstrate

ribopoly-nucleotides

their

protein

are

synthesizing

currently

ability

in vitro. --ACKNOWLEDGEMENT One of the

authors

Gobincl

Khorana

tory.

The authors'

Institute

for

for

gifts

'Shis work

explresses

providing

are

Servi:e

(CA-05178)

of Eiilcation,

of the

Dieter

and Grant-in-AfLd

for

in his

Physical

this

of Health,

U.S.

and

manuscript.

Foundation

Research

labora-

discussions

reading

Science

~perimental

H.

of Chemical

helpful

W. Moon for

Institutes

work

Suzuki

SE11 for

from National

National

to Professor

of this

Saburo

Malcolm

by grants

Institute

to do part

to Dr.

and to Dr.

was supported

Cancer

deep gratitude

als,o due to Dr.

of tubercidti, compounds,

Natio.lal

his

an opportunity

thanks

gift

of several

(M.I.)

(GB-3342), Public

(1965),

Health

The Ministry

Japan. SUMMARY

The stimulation synthesized

of the

trinucleoside

(jlj.5')-adenosine adencsine

binding

diphosphate

(TupApA),

(DMApApA),

analogs

with

thr-:RNA

to ribosomes

was significantly

tively.

No stimulation

was observed

type

residue.

hydrogen

These

by chemically

tubercidinyl-(3'45')-adenylyl-

TupCpC and DMApCpC was tested caused

to ribosomes

N6-d:iethyladenylyl-(~*+5~)-adenylyl-(j~+57)-

compared

adenine

that

of sminoacyl-sRNA

by other

results

bond formation

natural

by Nirenberg's

triplets.

The binding

stimulated

between

by TupApA

by the triplets

may best

codon

triplet

and

of lys-

and

and TupCpC respec-

containing

be interpreted

the

method

the

in terms and the

dimethyl-

of Watson-Crick anticodon

site

of sENA.

Acs,

G.,

Grif.?in, Ikehara,

Reich,

E.,

B. E.,

Ha&em,

M.,

Ohtsuka,

and Mori,

M.,

W. J.,

Proc.

Nat.

and Reese,

E. and Ishikawa,

Acad.

C. B.,

F.,

Sci., J. Mol.

Chem. Pharm.

j2, Biol., Bull.

493 (1964). l0,

353 (1964). 4, 173

(Tokyo),

(1961). Lohrnann, a.,

R., SXL, D., in press.

Hayatsu,

H.,

Ohtsuka,

263

E. and Khorana,

H. G.,

J.

Am. Chem.

Vol. 21, No. 3, 1965

Miz"~T9~;l, Nirenberg,

BIOCHEMICAL

Ikehara,

M., Watanabe,

M. and Leder,

Nirenberg, M., Leder, and O'Neal, C.,

P.,

AND BlOPHYSlCAL

K. A. and Suzaki,

Science,

l&5,

Sail,

A. and Davies, D., Ohtsuka, and Khorana,

1399

S.,

D.,

J. Am. Chem. Sot.,

Hayatsu, 73,

E., Jones, D. S., Lohrmann, H. G., Proc. Nat. Acad. Sci.,

Suzuki,

S. and Marumo,

Watson,

J. D. and Crick,

S.,

J. Antibiotics F. H. C.,

Toho,

Nature,

264

m,

J.

Orn.

Chem.,

a,3331

(1964).

P., Bernfield, M., Brimaxombe, Proc. Nat. Acad. Sci., a, ll61

Nishimura, S., Jones, D. S., Ohtsuka, E., H. G., J. Mol. Biol., U, 283 (1965). Rich,

RESEARCH COMMUNICATIONS

R., Turpin, (1965). H.,

3548

Jacob,

J.,

Rottman,

T. M. and Khorana,

(19.56).

R., Hayatsu, H., Nishimura, in press (1965). u,

F.,

360 (1960).

737 (1953).

S.