Role of modified nucleoside adjacent to 3t́-end of anticodon in codon-anticodon interaction

Vol. 40, No. 1, 1970

ROLE

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF MODIFIED

NUCLEOSIDE

ADJACENT

CODON-ANTICODON Kakoli

Ghosh

Department McMaster

Received

OF ANTICODON

IN

INTERACTION

and of

University,

TO 3'-END

H.

P.

Ghosh

Biochemistry

Hamilton,

Ontario,

Canada.

May 26, 1970

Summary: Removal of the base Y adjacent to the anticodon o yeast tRNAPhe changes its coding properties. Modified tRNA 6 he The codon UUU responds to the codon UUC better than the +5 odon UUU. can be read efficiently only at higher Mg cont. or in the presence of streptomycin. Binding studies show that the unmodified and the modified tRNAPhe can not occur next to each other on the two binding sites of a ribosome. Removal of Y changes the configuration of the anticodon loop of tRNAPhe such that the base (G) involved in "Wobble" changes its relative orientation to the other bases in the anticodon. The

phenylalanine

contain end

a flourescent of

the

adenosine case

of

tRNA

action.

of

any

and in

the

of

conformational

or

removal

nucleoside it

is

structure

(1,2).

Occurrence

a modified

nucleoside

a possible

role

(5)

have

position

is

the

than

is

needed

codons

the

Previous

base

to

essential

for

binding for

been

of

prevent

charging

anticodon

of 135

the

this

the

U or

A

in

reading may

also

has

the

(6,7,8).

the

the

increase

anticodon

suggest

of by

the

involving

the

inter-

function

ribosome-mRNA tRNA

3'-

in

position

the

It of

to

the

a modified

with

the

studies

tRNA

to

observed

that

anticodon

the

germ

codon-anticodon

anticodon.

flexibility. adjacent

of

the

conformation of

wheat

adjacent

at

in

to

and

beginning

proposed

the

stacked

yeast

have

of

("Wobble@'base)

the

anticodon to

other

of

not

unknown

of

) of

corresponding

this

of

5'-base

Y of

Hodgson

triplet

stabilization

that

(tRNAPhe

3'-end

suggests

purine

codon

the

presence

Fuller

alkylated

but

to

loop

RNA

2'-OMeGAA

species

The

anticodon

base

anticodon, next

(3,4).

the

transfer

such

maximal

degree

modification that

this

complex

Vol. 40, No. 1, 1970

The

results

the

presented

base

cont.

in

the

removal the

of

anticodon

the not

Yeast

acid

coated

with

The for

1.2

rations

of

pH

buffer,

described

isolated by

1.1

the

T and

G were

a procedure separation

Mg +2

a higher

the

suggest

that

configuration

loop.

The

conformation

tRNAFhe

and

5'-base, and

and

2'-OMeG

therefore

from

aminoacids --et

were

using

al.

described

Nishimura

2.

___ coli

MRE600

either

of

Erbe

--et

(16).

involving followed

al.

from the by

both

--et by The

E.

-coli

separation

partial

tRNAGhe

per

A260

as

assayed

separation

136

0.05M

of

flourescence

cacodylate

tRNA

species

synthetase

as

was

purified

Gordon

E.

acids the

prepa-

were (15)

polymerizing and

nucleic

could

Ribosomes

aminoacid

of

the

synthetase

method

two

260 pH 2.9,

The

by

(14).

the

at

isolated

aminoacyl-tRNA

al.

A

the

chromatography

The

MRE600 of

acid

unit.

in

Aminoacyl-tRNA

(13). by

purified

The

(11,12).

yeast

silicic of

per

with

BD-cellulose

determined

MgC12

on

phenylalanine

by

tRNAPhe

(BD-

activities

tRNAPhe

phenylalanine

0.015M

acceptor of

(7).

spectra

containing

The

isolated

- cellulose

chromatography

treating

Zachau

free

DEAE

further

nmoles

by was

were

Ghosh

benzoylated by

1.3

removed

Thiebe

with

method

by

to

tRNA;he

from

its

preferentially

results

changes

rigid

(9,lO).

of

as

a more

followed

and

7.0

yeast

anticodon

isolated

nmoles

by

of

the

at

of

U.

1.5

charged

from

was

Flourescence

spectra.

only

removal

affects

UUC is

These loop

with

Y was

by to

recognized

anticodon

have

were

4 hours

accept

codon

streptomycin.

BD-cellulose

base

described

be

the

the

tRNA Phe

yeast

cont.

that

Methods:

isolated

unit.

were

now

chromatography

tRNAPhels

as

the

pair

tRNAPhe

cellulose)

of

show

of

Mg +2

pattern

"Wobble"

and

anticodon

UUU can

Y from

may

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

communication

low

codon

stacking

Materials

the

presence

of

hence,

this

At The

or

37O

to

properties.

recognized.

can

in

Y adjacent

coding

the

BIOCHEMICAL

coli by factors

or factor

B cells polymer

by phase

T and

G

Vol. 40, No. 1, 1970

by

(NH4j2

on

Polypeptide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(15).

The

a DEAE-Sephadex

column

synthesis

ribopolynucleotides

sequences

were

ribosomes

in

was

fractionation

SO4

purified

BIOCHEMICAL

followed

using

followed

as

as

before

the

peptidyl

site

as

described

by

two

described

(13).

--et

were

and

al.

further

by

Erbe

of

repeating

Binding

(P site) Ono

factors

of

--et

al.

(16).

aminoacyl-tRNA

aminoacyl

site

to

(A site)

(17).

Results: It

was

previously

transfer both

shown

phenylalanine poly

r-U

and

Results

(18). Phe-tRNAihe

that into

poly

to

transfer

tRNAPhe

charged

polyphenylalanine

r-(U-U-C),

presented

fails

yeast

in

under

using Figure

homologously the

a ribosomal

1 show

phenylalanine

that into

direction

system at

can

a Mg+2

from cont.

polyphenylalanine

of --E.

coli

of

1OmM as

A A-

-C-OMe

A G-Ok U C -0Me

/A ,G ,A ,C ,c

G

ll

G A. Fig.

1.

G Phe tRNA

B.

c-

I Phe tRNAp

Kinetics of incorporation of phenylalanine. The reaction mixture contained per ml: Tris-Cl. pH 7.5, 10 nmoles; NH4C1-160 nmoles: DTT-10 pmoles; GTP-1 washed ribosome-21 A260 units; umole; T and G factor C14-Phe-tRNAPhe-350 pmoles or C14-Phe-tRNAThe-362 - 48Oug; 'nmoles and Mg acetate as indicated. The template cont. was 1.0 A260 units of poly U or 5.8 pmoles of poly r-(U-U-C) Phenylalanine polymerized was assayed as described per ml. earlier (13). 137

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i

c 8 N

138

BIOCHEMICAL

Vol. 40, No. 1, 1970

directed

by

poly

Phe-tRNAPhe triplet

and

the

these

by

75

of

poly

of

fold.

is, that

of

in

1.

Table

effect

on

cont.

from

Presence

of

of cont. that

anticodon

loop

longer

pair

A steric

enzymatic

P site.

of

with

of

between

on

thus the

binding

C14-Phe-tRNAPhe

the

occur

Phe-tRNA

N-acetyl

results Y

changed

can

are not

they

shown has

1OmM and

20mM Mg

no

poly

r-U

by

25-fold.

At

The

+2

results

loop

relative

in

above

anticodon

tRNA

molecule

higher

tRNA Phe

of

loop,.

The

orientation base

occupy

the

complex.

Phe at the Y Phe-tRNAPhe

to

are

by

"Wobble"

modified when

Phe-tRNA;he

10 ug/ml

anticodon the

the

in

(2'-OMeG)

can

the no

cont.

presented bind

the

its

the

UUC are

Polyphenlalanine

observed.

of

ribosome-mRNA

of

containing The

have

both

stimulated

configuration

presence

studied

results

r-(U-U-C).

Y from

tRNA Phe so that Y U at lower Mg+2

may

sites

the

base

we

of

at

is

r-U

UUU and

from

directed

however,

poly

These

(19),

a cont.

poly

stimulates

in

codons

The

at

by

tRNA;he

tRNA;he.

r-(U-U-C).

Phe-tRNAFe

the

might

hindrance

molecule

binding

from

by

Z-fold.

the

phenylalanine

directed

20mM

incorporation

stimulation

in

2'-OMeGAA

miscoding

poly

is,

removal

anticodon

induce

streptomycin

significant

a change

of

of

streptomycin no

produces

and

by

both

phenylalanine

incorporation

Phe-tRNAPhe, Y at 1OmM Mg +2

presence

suggest

r-U

cont.

anticodon to

on poly

the

synthesis

+2

both

the

known

streptomycin by

complex

by is

Mg +2

to

Phe-tRNA;he

only

the

of

directed

from

both

Clearly

cont.

Phe-tRNAGhe

increased

by

(2'-OMeGAA)

Mg+2

synthesis

higher

efficiently

directed

tRNA

at

recognized

condition.

the

from

however,

is

anticodon

Increasing

streptomycin

effect

the

Polyphenylalanine

recognized

however,

identical

by

phenylalanine

suggest

Since

recognized

r-(U-U.-C)

results

Mg

not

under

conditions.

transfer

r-(U-U-C),

Phe-tRNAGhe

UUU is

under

Poly

r-U.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in poly

or

the

therefore,

of

r-U-ribosome

2.

The

normal

consecutive

poly

tested

at

results complex

the

r-U-ribosome

N-Ac-Phe-tRNAGhe

Table

139

two

We,

A site

and

the

show at

both

that 1OmM

20 mM

10 mM

+2 % cont.

Ac-H3-Phe-tRNA

bound

of

Phe Y

(p

to

22.0

11.0

14.0

5.4

moles/ml)

cl4 -p;;:;;NA;e

C 14-Phe-tRNA

2

bound

A site

20.3

< .05

7.8

< .05

Ac-H3-Phe-C14-Phe formed

The two step reaction as described by Ono --et al. (17) was followed. In step I the reaction mixture contained per ml. of ribosome 4 A260 units of poly U, 126 31 A260 units Phe or 135 pmoles pmoles of Ac-H3-Phe-tRNAp of Ac-H3-PhetRNAPhe and Mg acetate as indicated. Step II reaction mixt 8 re contained per ml.0.05 pmoles of GTP, 1 pmoles of Fusidic acid, 150 l.lg of T, 435 pmoles of C14-Phe-tRNAshe and Mg acetate as indicated. Both reaction mixture contained 0.04M Tris-Cl, pH 7.4, 0.01M DTT and 0.16M NH4Cl.

23.6

72.0

Ac-H3-Phe-tRNAPhe

71.2 7.0

Phe v

Phe

Total Ac-H3-Phe-tRNA

-Phe-tRNAye

Ac-H3

Ac-H3-Phe-tRNA

Ac-Phe-tRNA in the P site

'Binding

TABLE

s.-

BIOCHEMICAL AND BIOPHYSICAL

Vol. 40, No. 1, 1970

(Mg++)

(M9 ++)

= 1OmM

RESEARCH COMMUNICATIONS

=2OmM I

k“? _ c+Pdv

15

30

Phe-1RNAPPs u v

60

90

1

Time [min] Fig.

2.

Schematic diagram of the model for tRNA anticodon arm. (a) Fuller and Hodg son model for tRNAPhe. (b) Proposed alternate model for tRNA:he. The alternate model proposes a change in the stacking pattern of the anticodon only. The helical character of the structure is considered to be the same as postulated by Fuller and Hodgson (5).

20mM Mg +2 when the Phe tRNA . On the other and

P site

the

ribosome

when

the

P site

is

occupied

by

ribosome-poly Phe Phe , C 14 -Phe-tRNA can bind is occupied by N-Ac-H3-Phe-tRNA Y Y 14 -Phe site and the dipeptide N-Ac-H3-Phe-C is synthesized. strongly of

suggest

the

loops

anticodon of

tRNA

hand

of

tRNA Phe

that loops.

Phe

and

and

tRNAye

is

of

have

tRNAthe

A possible

different

schematic

diagram

in

2.

shown

N-Ac-H3-Phe-

Figure

r-U

complex

to

the

These

A results

configuration of

the

anticodon

Discussion Previous to

the

workers

anticodon

reported or

modification

that

removal of

the

141

of

the

side-chain

modified of

base this

base

adjacent affects

Vol. 40, No. 1, 1970

its

binding

to

transfe.r

BIOCHEMICAL

capacity

to

aminoacids

however,

show

of

codon

recognition

of

the

by

anticodon

are

now

no

longer

the

flexibility

the

Mg

of

the

known

to

tRNA

molecule

Y however, Also GTP

and

base

and

Phe

the

(unpublished

Fuller

and

more, is

our

results

possible

and

alternate

to

as

streptomycin

the

known

of

codon

as

the

is

so

of

that

5' -base

show

that

the

alternate

active.

anticodon

conformation

stacking

an

functionally

studies

in of

loop of

of

is

loop,

base

anticodon

Phe

of of

have

Y the

> with modified

recognized

and

may

model

pairing.

support

the

is

anticodon

not

structure of

cont.

The

that

lend

Results tRNAiet

+2

(12,21).

normal

loop

loop

the

the

the

, indicating

anticodon

involved

tightens

(T-GTP-Phe-tRNA-

anticodon Our

stability

loop

involves

Phe

Increasing

Mg

of

has

the

Also

anticodon

nature

the

base

increase

configuration

Phe-tRNA

and

pairing.

(20).

the

conformation

"Wobble"

streptomycin

ability

finding,

only

the

The

complex

its

pyrimidines

may

UUC which

of

affects

"Wobble"

the

tertiary

well

model

its

miscoding

changed

the

two 2).

that

observation).

base loop

binding

forms

Hodgson

modified

anticodon

of

configuration

factor

the

the

that

for

allow

the

base

(Fig.

conformation

with

TT T equally

such

hence, Our

Presumably,

needed

is

and

(6,7,8).

modified

together

and

recognizes Phe-tRNA

changed

It

the

this

chain

anticodon.

presence

ribosome

change

Phe

is

pair.

of

the

stacked

or

"Wobble"

structure

of

RESEARCH COMMUNICATIONS

complex

polypeptide

removal

loop

cont.

mRNA-ribosome

into

that

anticodon

+2

the

AND BIOPHYSICAL

by

to

the

suggest

bases

in

that the

"Wobble".

Further-

anticodon

loop

conformatio

oligonucleotide

already

suggested

the

an

anticodon loop (22). for his interest and critical We thank Dr. R. H. Hall Acknowledgment: This work has been supported by a grant from the Medical discussion. is a Medical MA 3514 ). H.P.G. Research Council of Canada (grant no. Research Council Scholar. References 1. 2.

u. L. RajBhandary, S. H. Chang, A. Stuart, and H. G. Khorana, Proc. R. M. Hoskinson, U.S., 57 751 (i967). B. S. Kdock, G. Katz, E. D. Taylor, and Natl. Acad. Sci., U. S., 62, 941 (1969) 142

R. D. Faulkner, Natl. Acad. Sci., R.

W.

Holley,

T

Proc.

Vol. 40, No. 1, 1970

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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143