Conformation of N4-acetylcytidine, a modified nucleoside of tRNA, and stereochemistry of codon-anticodon interaction

Vol. 83, No. 2, 1978 July 28,1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 657-663

CONFORMATION OF N4-ACETYLCYTIDINE, A MODIFIED NUCLEOSIDE OF tRNA, AND STEREOCHEMISTRY OF CODON-ANTICODON INTERACTION

R. Parthasarathy*t,

Stephan L. Ginellt, and Girish B. Chhedatt

Nimai C. Dett

Center for and Center Roswell

Crystallographic Researchttt for Developmental Oncology Park Memorial Institute 666 Elm Street Buffalo, New York 14263

Received

June 8,1978

The crystal structure of N4-acetylcytidine (ac4C) a modi ied nucleoside =F: of tRNA, has been determined from threedimensional x-ray diffractometer data. is The N4 -substituent proximal to C(5), quite contrary to expectations from solution studies of N4-methylcytosine. This orientation of the N4substituent will not block Watson-Crick base pairing for reading the third codon by tRNA!et, and hence the discriminatory function suggested for ac4C might arise due to non-standard conformation of the polynucleotide backbone of the anticodon around the Wobble base. A common characteristic of the modified nucleosides that occur at the wobble position is their inability to shield the Watson-Crick base pairing sites; this is quite consistent with the necessity for reading the third base of the codon. This is in sharp contrast to the modifications of the nucleosides adjacent to the 3'-end of anticodons, all of which prevent Watson-Crick base pairing.

Introduction: that

occurs

tRNAFt

N4-acetylcytidine,

ac4C,

the

of the

at

(1,2),

first

position

and in the dihydrouridine More recently,

rRNA and appears

to be conserved

(6).

Examining

nucleosides

adjacent

(7-10)

irrespective

the

that

inability

pairing

(11)

to the

to base pair is

of

a modified

anticodon

nucleoside

of E.

C&

stem in tRNASer

(3,4)

ac4C has been identified

and tRNALeU (5).

of eukaryotes

is

in the

the

ribosomal

stereochemistry

3'-end

of anticodons,

the type according

a common feature

small

of chemical

by all

subunit

of modified we have found modifications

to the Watson-Crick

shared

in 18s

base

these modified

0006-291X/78/0832-0657$01.00/0 657

Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol. 83, No. 2, 1978

nucleosides.

On the

nucleosides ('Wobble' ing;

BIOCHEMICAL

that

other

occur

position

the C(4)-N(4)

on the

The 'distal'

base pairing

while

If

then

modified

ing for

this

tRNAFt

code will

would

appeared

pairing

the results

of of our

of

preference

will

standard

N.m.r.

distal

part

than

studies

important

on the

ac4C codon read-

and the

three

letters

(10).

and this

crystal

results

for

in the

of ac4C to G must be used

ac4C

conformation

base pairing,

as two rather

of

Watson-Crick

prevails

take

across

ability

not.

also

cannot

base pair-

prevent

the

conformation

anticodon

group

base pairing

for

on

to be an exception

one will

ac4C is very

the

of the N-acyl

'proximal'

the

the modifications

Watson-Crick

conformation

to be read

stereochemistry

genetic or a

Hence, paper

structure

the

describes

and conforma-

of ac4C.

Methods.

The nucleoside

fications

of known procedures

ac4C was synthesized

from water-propanol

orthorhombic,

space group

a = 18.177(l), Dtalc.

= 1.57

flections

g*cmw3.

to the

limit

Three

a GE XRD-6 diffractometer by the multisolution

R of

using

0.051

approximation.

the

2 = 4,

intensity

technique

hydrogen

maps; their

difference

658

Do&d.

data

modi-

are (22 + 3)'C: = 1.57,

(1577 re-

were collected and the

(16) and refined

method with

of the

at

CuKcc radiation)

and Ross filters

least-squares

The locations

from electron-density

constants

2 = 5.692(1)i;

20 = 165O for

was solved

The crystals

cell

dimensional

slight

of ac4C (CllHl5N306)

solutions.

P212121 with

b = 11.650(l),

using

Crystals

(15).

were obtained

using

prevent

the

nucleoside

RESEARCH COMMUNICATIONS

position

orientation

distal

using

have

non-standard

tion

the a 2O:l

(13,141. this

first

bond of cytosine,

m4C indicate

none of

however,

is altered.

for

the

(12)),

Depending

(10).

hand,

at

N4-acetylcytidine,

AND BIOPHYSICAL

the

structure to an

block-diagonal

atoms were obtained positional

parameters

BIOCHEMICAL

Vol. 83, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H C8) H2bl HllCI)

c(8)

b

r

A a13

Conformation Figure 1. substituent on N(4) is tion across C(4')-C(5')

and individual

isotropic

of N*-acetylcytidine. oriented proximal is gauche-.

thermal

Note that the The conformato C(5).

parameters

were included

in the

refinement.

Results

and Discussion.

the molecule.

Figure

The 'proximal'

1 illustrates conformation

the conformation of the molecule

with

of

Vol. 83, No. 2, 1978

the

substituents

N(3)

and O(2)

in

spite

BIOCHEMICAL

on N(4) to take

of

with

is

pairing

trans

in

of m4C.

Since

was felt

that

Further,

model

building

this

the

G, anti

C(Z')-endo

bond

is not

the

conformation There

is

hydrogens

no base

take

part

in

the

but

structure

the

electron

probably

has polarized

hydrogen

bonding

proton

downfield

to 7.25

proton.

lack

it

it

be distal. from

Consequently, H(C5)

126O)

to O(7)

in

the

contact

crystal

leave

H(C5)***0(7)

giving

rise

to

the

conformation

(C(7)-N(4)-C(4)-C(5)-H(C5)***0(7)). to note

hydrogen

withdrawing

Our n.m.r.

studies

660

to

of

repulsion

6.04

take

proximal

this

part

that for

of

acidic

group in

the

C moved acidity

has been

acetates

the

more in

indicate

an increasing

of that

(17-19);

sufficiently

ac4C indicating

the

of N-acetyl

has a 6 value

of H..*O=C

that

bonding ability

also

possible

conformation

contacts

intramolecular

conformation is

short

un-

group,

of ac4C will

is

interesting in

usually

preferred

Thus,

the

H(C5)

for

Also

the

is

(19).

which

angle

ring

part

a methyl

orientation.

attractive

six-membered

takes

than

and the

***O(7)

it

on the

indicated

proximal

is quite

(13,14)

conformation

to regard

connection,

N4 -substituent

bulkier

conformation

repulsive

usually

is

studies in

but

of a planar

(20,21).

with

has the

gauche-

polar

results

group

(C(5)-H(C5)

as not

explain

ring

to C(3')).

of the

preferred

proximal

2.15(5)ii

this

H(N4),

exhibits

C(4')-C(5') the

All

n.m.r.

an acetyl

acetyl

observed

H(C5)

bases.

of the

the

to the

H(C6)

but

orientation

view

no alternative

In

the

enable

bonding.

expected,

H(C5)

molecule

and -gauche

cytidine

will

base pairing

The sugar

conformation,

The proximal

tact

This

RESEARCH COMMUNICATIONS

C(5)

in Watson-Crick

across

to O(1')

of the

hydrogen

towards

xCN = 33.4O.

gauche+

(O(5')

of

part

The conformation

preferred

the

turned

the modifications.

conformation pucker.

AND BIOPHYSICAL

used

for to

cyclohexanols conformation

Vol. 83, No. 2, 1978

8lOCHEMlCAL

observed

in the crystal

solution

and could

prevail

that

functional

role

the

aimed at preventing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is also

the

preferred

in tRNA also.

The modified

nucleosides

the Wobble positions

Therefore,

of the N4-acetyl

the Watson-Crick

group

it

is

in appears

not necessarily

base pairing.

that

are Gm, I,

Conformation

have been found

"Q",

Cm, ac4C,

to occur

at

cmo5u, mcm5U,

mo5u, mnm5s2U and mcm5s2U (from

our compilation

of about

70 tRNA

sequences).

stereochemistry

of

nucleo-

sides

Examination

(10)

these

readily

shows that

modifications

is

base pairing

sites.

base pairing

is quite

the

third

inability

with

is possibly

to define

codon loop

for

the genetic

in E -*

coli -

and it

be related only

anticodon 3'-end

codon reads nucleoside anticodon comparison

to note

and an unmodified

as AUG) (22),

(2) that

in the

anti-

The presence specific its

of

the modi-

of anticodons

frame

(7,lO).

function

of

reading

for

tRNApt

uniqueness

tRNAFt

which

of

might decodes

AUG codon.

interesting

wobbles

for

3'-end

the reading

has been suggested

internal is

to the

ac4C seems to be quite

to the discriminative

the It

nucleoside

the Watson-Crick

the effect

code

all

the Watson-Crick

the necessity

adjacent

whose role

the modified

to shield

codons and unlike

on the nucleosides

of

to shield

inability

consistent

reading

these

a common characteristic

their

This

base of the

fications

of the

in the yeast

only

E. -- coli

adenosine

first

containing and E. -- coli

only

of the

the

association

ApUpG shows (25)

that

reduction

bonding

in the

internal

E. -coli

tRNAkt

of

with

to the antimodified

codon these

has the

which

661

on the

and t6A adjacent

constants

strength

to it

a CAU

(can read GUG as well

methionine

tRNApt

with

t6A adjacent

ac4C at the Wobble position reads

tRNApt

adjacent

codon position

tRNApt

AUG (23)

that

to the

(22,24).

three

lowest

seem to arise

A

tRNA with value. due to

The

Vol. 83, No. 2, 1978

modification the

of cytosine,

prevention

formations results

of for

the

these

gauche-

in

helical

gress

orientation

the

methionine

to evaluate

species

of

polynucleotide

chain

at

the

of methionine

Acknowledgements. This 24864 and CA-14185 from gratefully acknowledges

selective tRNA's

for

building

responses

of

Our

and a non-

level

the

con-

bond.

back-bone

be important Model

such as

ac4C.

C(4')-C(5')

polynucleotide

of the

tRNA's.

to

conformation

the

the

causes

adjacent

proximal

conformation will

several

ac4C to G or non-standard

across

persist

conformations

these

can be due to

ac4C has the

conformations

alteration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

base pairing

show that

standard

of

BIOCHEMICAL

from

If also,

the

selective

the

standard responses

studies

are

in pro-

these

different

(9).

work was supported in part by grants GMthe National Institutes of Health. S.G. a New York State predoctoral fellowship.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13.

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BIOCHEMICAL

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