Conformational differences between s-RNA and aminoacyl s-RNA

Vol. 20, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CONFORMATIONAL DIFFERENCES BETWEEN s-RNA ANDAMINOACYLs-RNA P. S. Serin and P. C. Zamecnik The John Collins Warren laboratories of the Huntington Memorial Hospital of Barvard University at the Massachusetts General Hospital, Boston, Mass.

ReceivedJune 22, 1965 Current interest been directed

in the mechanismof protein

biosynthesis has

to the study of the role of a-RNA,* m-RNAand ribosomes

in the biosynthetic

process.

According to present concepts, aminoacyl

s-RNA binds to the messenger-polysome complex, and after its amino acid to the growing polypeptide template.

To satisfy

transferring

chain, dissociates from the

the requirement of the binding of the anticodon

on S-RNA to the codon on the template, a conformation dependent parameter may be involved.

To search for a possible conformational

ence between s-R&! and aminoacyl S-RNA, the technique of optical

differrotatory

dispersion has now been employed. Optical

rotatory

dispersion measurementshave been widely used

in the past for the study of the helix-coil and proteins

transition

in polypeptides

(Urnes and Doty, 19611, and have been extended recently

to the study of polynucleotides and Tinoco, 1965; Lamborg-et al.,

and s-RNA (Fasmane al., 1965).

In an earlier

1964; Holcomb publication

(Sarin and Zamecnik, 1965), we reported on the application the study of

StNCtUrd

acceptor and transfer

of ORDto

patterns of the components of the amino acid systems. We now wish to report a conformational

-k Abbreviations: Tris, tris(hydroxymethyl)aminomethane; aminoacyl s-RNA, s-RNA charged with 21 amino acids; S-RNA, this polynucleotide stripped of all amino acids? m-RNA, messenger RNA; A, adenine: C, Cytosine; G, guanine; 0, uracil; and ORD, optical rotatory dispersion.

400

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 4,1965

difference

between

mined whether binding

such may be related

to the m-RNA-ribosome

unesterified

stripped

amino acids

mixture

4 vols.

was cooled

of absolute

was dialysed

ing of s-RNA. material (Scott,

incubation, with

0.1 M sodium chloride

with

was repeated

respect

reported

and aminoacyl

(3:1,

was well

for 2 hrs.

dispersion

(Lamborg et al.,

s-RNA were

until

determined

of protein

v/v)

shaken,

2 ~01s.

and the

The extraction there

to sodium chloride

was no prethe aminoacyl

and dialyzed

and finally

The dialyzed

and the sample was checked for rotatory

and Zamecnik,

a-EUVAwas freed alcohol

The mixture

a Gary 60 spectropolarimeter

already

(Sarin

follow-

(r4C Phe) was added to check the label-

in the cold overnight.

Optical

Water

lyophi-

21 amino acids,

The aqueous phase containing

(aqueous)

extrac-

water.

and finally

reported

chloroform:isoamyl

alcohol

s-RNA was made 0.1 M with

lyophilysed,

by adding

changes the sample

was removed by centrifugation.

at the interface.

water

for 48 hrs.

the

dissolved

distilled

three

the aminoacyl

procedure).

chloroform:isoamyl

distilled

the first

water

C amino acid

precipitate

cipitate

and after

against

to the ones already

by extraction

separated with

14

unpublished

incubation,

Tris was removed by this

s-RNA was then charged with

After

After

1964).

of Tris-

and s-RNA was precipitated

The excess

distilled

similar

One marker

Ohio) was

at 37 ' in the presence

and dialyzed

2 hre.,

against

ing conditions

Falls,

s-RNA was removed by centrifugation,

The stripped

1965).

degree of

s-RNA compared to

Chagrin

and Zamecnik, in ice,

in a small volume of water, was changed every

Biochemicals,

ethanol.

The precipitated

lyzed.

to be deter-

greater

of aminoacyl

by incubation

(1.8 M, pH 8) (Sarin

reaction

It remains

to the apparently

complex,

s-RNA (General

of all

acetate

s-RNA.

s-RNA.

E. coli

tion.

s-RNA and aminoacyl

dialyzed

against against

sample was then

the absence of ATP.

was measured from 320 mp to 230 mp using 1965).

conditions

similar

08D measurements

at a concentration

401

to the ones of s-RNA

of 0.4 mg/ml

Vol. 20, No. 4, 1965

BIOCHEMICAL AND BlOPHYSlCAL RESEARCH COMMUNICATlONS

in 0.2 M sodium was determined

citrate by inorganic

s-RNA and aminoacyl in order

buffer

to gain

stabilities

(pH 7).

phosphate

s-RNA were insight

Concentration

into

to protonating

also the

of a-RNA samples

estimation. carried

OlUJ measurements

out as a function

similarity

of

of pH,

or differences

in their

environments.

-41 220

240

260

260

300

320

WA YEa! ENGTH, tq~

Fig. 1. Optical rotatory buffer. pH 7, (0 -0)~ (X -8); pH 3, (U--a>.

citrate 3.5,

As can be seen fran Cotton

effects

is observed

90% of the amplitude of the from

ordered

but

No change

on lowering

due to the protonation

the Cotton

effects

amplitude tude

becomes

concomitant relatively changed

from

3.5

change,

about

to 3.

disruption

the pH ie- changed occurs,

The amplitude

of

at pH 7.

Further

a considerable

change,

and the

at pH 7.

change

in ampli-

‘Ibis

of the A residues bon&d

57% to 49%, is

with

structures.

observed

Thue at pH 3 when most of the

402

of

to be

disruption

of the amplitude

of the A-U hydrogen from

when

amplitude

is found

a partial

of the C residues.

due to the protonation

disruption small

thereby

the pH to 4 a further

57% of the amplitude

ia presumably

in the

amplitude

is observed

at pH 4 ia 84% that

of the pH to 3.5 brings

s-RNA in 0.2 M aodium pH 4, (r--q>; pH

change

at pH 5, when the

presumably

lowering

1 the first

at pH 7, suggesting

structure.

5 to 4.5,

Fig.

dispersion of pH 5, (O---O);

when the

a Only a pH is

C and A residues

BIOCHEMICAL AND BlOWlYSiCAL RESEARCH COMMUNICATIONS

Vol. 20, No. 4,1965

would have been protonated,

the amplitude

found to be SD% of the amplitude to longer wavelength the pH is lowered that

this

from 4 to 3 through

structures

suggesting

which

seem to shield

regions

hydrogen

likely

in strong

to be disrupted

suggest rather

that

This suggestion alanine

agrees with

s-RNA by Holley

2.

in the secondary

82% of that lowering

of s-RNA.

is also

decrease

observed,

nitrogenous

cific

bonded helical

to

regions structure.

determination

of

(aminoacyl

seems to occur, effects

of aminoacyl

A large decrease

with

with

again suggesting

s-RNA is respect

in amplitude

to the change observed

in amplitude

with

of differently

to

occurs s-RNA.

change in pH of aminoacyl

the presence

an

as shown in Fig.

change in amplitude

is parallel

s-RNA),

s-RNA

shielded

bases.

In order ference

ordered

structural

of Cotton

at pH 4.5.

at pH 3.5 and 3, which

are

seems plausible

21 amino acids

A noticeable

of pH occurs

A non-linear

Thus it

which

(1965).

structure

Thus at pH 7 the amplitude

The non-linearity

bonded regions

hydrogen

When s-RNA is charged with alteration

of the bases

could also be due to the engulf-

the primary

--et al.

ordered

of the nitrogenous

is composed of short,

completely

in pH

of different

bonded environments.

only at low pH.

than a single,

lowering

of the bases in hydrophobic

GC hydrogen

s-RNA structure

to note

The inaccessibility

in the opening of the s-RNA structure ing of some bases

with

the availability

environments.

occura when

is of interest

of regions

may be due to the burying

or in strongly

It

is

a marked shift

point

structure

the presence

baaea to the protonating to protonation

3.5.

of the secondary

effects

In addition,

at pH 7.

of the peak and the crosswer

disruption

is non-linear,

of the Cotton

to answer

in amplitude absorption

the question

might be simply

as to whether

a reflection

at 260 mt~, a sample of aminoacyl

at 37O in the presence

of Tria-acetate 403

(2 M, pH 8).

the observed

dif-

of a change in apeS-RNA was incubated Fifty

~1 aliquota

Vol. 20, No. 4,1965

BIOCHEMICAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS

+4

“:

t2

9 z 0 2 4. -2

-4 220

240 260 WA YEL ENGUf,

280 m/

300

320

Fig. 2. Optical rotatory dispersion of aminoacyl M sodium citrate buffer. pH 4.5, (o---O>; pH 7, (0 -0); (V-r>; pH 3.5, (X-X); pH 3, (CI--0).

containing

40-50 ng s-RNA were

to 1 ml with

shift

ultraviolet

the radioactivity Both types

spectrophotometer.

was observed due to

over a period 14

of the aminoacyl

alcohol s-RNA.

at 260 mp was then

of 90 min.

during

and dialysis

The difference8

and the pH readjusted It becomes apparent

charged with

21 amino acids

compared to s-RNA strfpped conformation binding

of aminoacpl

of aminoacyl

this

All period.

of incubation,

during

preparation

in the ORD of aminoacyl

and s-RNA were also found when a sample of aminoacyl dialyzed

hypo-

incubation.

to the same procedure

extraction,

and diluted

no detectable

C amino acid was lost

of sample were subjected

chlorofozm:isoamyl

intervals

The absorbance

0.14 M sodium chloride.

read in a Zeiss chromic

removed at 10 min.

s-RNA in 0.2 pH 4,

a-RNA

s-RNA was stripped,

to pH 7. from the above observation6 has a different

of all

conformation

amino acids.

s-RN4 may be important

e-RNA to the template

404

that (less

s-RNA helical)

This difference

in the

in the preferential

in protein

biosynthesis.

Vol. 20, No. 4, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgments The authors are helpful criticism. This Cancer Society (II-102G) AT(30-11-2643). This is of Harvard University.

indebted to Dra. J. F. Scott and M. Lamborg for work was supported by grants from the American and the Atomic Rnergy Commission (Contract No. publication Wo. 1213 of the Cancer Commission

References Fasman, G. D., Lindblow, C., and Groasman, L., Biochemistry, 3, 1015 (1964) Holcomb, D. N., and Tfnoco, I., Biopolymere, 2, 121 (19653 Holley, R. W., Apgar, J., Everett, G. A., Madison, J, T., Marquisee, M., Merrill, S. H., Penwick, J. R,, and Zamir, A., Science, 147, 1462 (1965) Lamborg, M. R., Zamecnik, P. C., Li, T. K., Kagi, J., and Vallee, B. L., Biochemistry, 2, 63 (1965) Sarin, P. S., and Zamecnik, P. C., Biochfm. Biophya, Acta, 9l, 653 (1964) Sarin, P. S., and Zamecnik, P. C., Biochem. Biophys. Res. Conmt., l9* 198 (1965 > Urnes, P., and Doty, P,, Adv. Protein Chest., l6, 401 (1961)

405