A specific spin labeling of the anticodon of E.coli tRNAGlu

Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A SPECIFIC SPIN LABELING OF THE ANTICODON OF E. COLI tRNA Glu.

A°R. Mclntosh, M. Caron and H. Dugas

#

Department of Chemistry Universit6 de Montreal, Montreal i01. Canada. Received

November

6,

1973 SUMMARY

The rare base 2-thio-5(N-methylaminQ~ethyl)-uridine (seu *) in the of the antlcodo _~i~ I_ . ~ wobble p osltlon " " " n of E. ~ . . . .~ ~, ^ ~ ± u ~Las been specizically acylated with the mixed anhydride spin label I. A temperature-induced conformational study showed a transition at 50 °. These results are discussed in terms of accessibility and stability of the anticodon region and are compared to previous works reported in the literature.

INTRODUCTION Considerable efforts have been devoted to an understanding of the various functions of transfer RNA's I-4. given the most exciting result.

A recent x-ray study ~ on tRNA Phe has

Furthermore, h i g h r e s o l u t i o n PMR studies 6-8 and

spin labeling studies 9-12 have been of great importance as well.

However, in

order to obtain precise structural informations about the environment and stability of a particular region of a tRNA molecule, it is convenient to be able to introduce a reporter group at a specific site on the macromolecule.

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Copyright ©1973 by Academic Press, lnc. All rights o f reproduction in any form reserved.

il 0 C2 H 5

Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We report here preliminary results of an investigation

of the thermal

behavior of the anticodon region of glutamic acid specific tRNA from E. coli labeled with the mixed anhydride spin label I.

Enzymatic hydrolysis

showed that

this paramagnetic probe specifically acylated the rare base 2-thio-5(N-methylaminomethyl)-uridine

(s2U *) which is part of the anticodon of this tRNA.

Cedergren et al. ~3 reported an identical reaction using radioactive

Recently, aromatic

anhydrides.

MATERIAL AND METHODS Glutamic acid specific tRNA from E. coli K-12 (iot.15-291) (activity 78%) was kindly provided by Dr. A. Kelmers of the Oak Ridge National Laboratory. Bentonite was obtained from Canadian Foundry Ltd. (Montreal) and was purified according to Fraenkel-Conrat et al. 14 DEAE-cellulose was purchased from SchwarzMann and bovine pancreatic ribonuclease A was a product of Sigma Chemicals. The spin label anhydride I was prepared according to the method of Griffith et al. Is, m.p. 65°-67 ° (litt. 65o). All other chemicals were reagent grade or of the purest form available. Spin labeling of E. coli tRNA Glu. The reaction was performed using a Metrohm pH stat loaded with 0.I N NaOH in a 1 ml microcell thermostated at 25 ° . To a solution of tRNA Glu (max. of 6 mg) in 1.0 ml of deionized doubly distilled water at pH 8.0, containing i0 mM MgCI2 and 3% bentonite, to protect the system from ribonucleases, was added the solid mixed anhydride spin label I (max. of 4 mg). The reaction started immediately and was judged completed after two hours. In order to separate the unreacted spin label from the tRNA, the reaction mixture was chromatographed on a Sephadex G-25 (coarse) column (70 X 1 cm) using a 0.02 M Tris-HCl buffer (pH 7.5) containing i0 m M M g C I 2 as eluent. The elution was monitored at 260 nm. The resulting fractions containing tRNA were combined, dialyzed in a Bio-Rad minibeaker at 4 ° and then lyophylized. The resulting solid material was stored at -20 °. Attempts to separate the labeled from the unlabeled material have been so far unsuccessful. For EPR measurements, this material was dissolved in 0.i ml of a 0.02 M Tris-HCl buffer (pH 7.5) containing i0 m M M g C I 2 . Digestion of tRNA Glu with bovine pancreatic RNase A. A complete pancreatic digestion was carried out using the procedure of Cedergren et al. (private communication). The resulting digest was then applied directly on a DEAE-cellulose column (120 X 0.5 cm) eluted with a concave gradient of ammonium carbonate 13. All the fractions comprising peaks of significant optical density at 260 nm were collected together and were lyophylized to near dryness. The individual samples corresponding to peaks in the elution pattern were then taken up in 150VI of deionized doubly distilled water for spin labeling detection by EPR.

Measurements

of EPR spectra.

The EPR spectra were recorded on a Bruker 414S Spectrometer operating at 9.5 GHz and equipped with a rectangular cavity. The microwave power was kept below 5 raW. The samples were taken into a small aqueous flat cell (Scanlon Co.). For variable temperature studies, a Bruker temperature control unit was used and the temperatures were accurate to better than T 1 °. Rotational correla-

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Vol. 55, No. 4, 1973

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

tion times (Tc) from EPR spectra were calculated using the formalism of Kivelson 16 according to Waggoner et al. 17 The T c values reported here were obtained from the term quadratic in the nitrogen nuclear quantum number.

RESULTS

The cloverleaf structure of E. coli tRNA Glu based on the sequence determined by Munniger and Chang 18 is depicted in Figure i.

t.RNAGlU

The spin labeling re-

Ac

Coli

c G G--C U--A C--G C--G C--G C--G U~A

G

A

A

A

U

~

G

UC

II

C CUG

| i

G

GAC C

AGG

|

A C

C A

C

G

G

U

A

T

C

I I I

e

A

U

C

A

C C

C~G C--G G~C C~G C~G C

C

U

m~A

sLU ~

C U

/

Fig. i. Cloverleaf structure of E. coli tRNA Glu. site of labeling.

The arrow indicates

action was carried out in an aqueous medium containing i0 m M M g C l z is presumed to assume its native conformation.

where the tRNA

A spin label concentration determi-

nation 19 showed that about 8 to 12% of the total tRNA Glu was spin-labeled. the yield of the reaction was moderate, pancreatic RNase digest

the

Although

the elution pattern issued from the total

(Fig. 2) indicated that almost 90% of the total EPR signal

level was contained in one peak.

The position of this peak in the elution profile

was found to be consistant with that of Cedergren's benzoic anhydride radioactive product from a similar DEAE-cellulose

chromatography 13.

Since these authors have

performed their acylation reactions on tRNA Glu using anhydrides as coupling agents, it is pratically

certain that our acylated tRNA Glu is labeled in a similar way.

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Vol. 55, No.4,1973

0.30

LO 5

0.20 M

>

O.lC

g M

106 O.OC 0

125

250

375

500

(ml) Fig. 2. DEAE-cellulose elution profile of the complete RNase digest of labeled and unlabeled tRNA Glu. The shaded zones represent the relative amount of spin labeled material collected from the elution.

That is, our reaction also achieved the specific labeling of the rare base s2U*at the wobble position of the anticodon of this particular tRNA. @ Figure 3 shows the EPR spectra of the spin-labeled spin-labeled nucleotide isolated from the DEAE-cellulose the RNase A digestion.

As expected,

chromatography

following

the latter material gives a spectrum

typical of a spin-labeled mononucleotide 21. spin-labeled

tRNA Glu and of the

However,

(Fig.

3b)

the solution containing the

tRNA Glu yields an EPR signal of intermediate mobility

(Fig. 3a) indi-

cating that the motion of the covalently bound spin label is to some extent impeded by the macromolecule,

A quadratic rotational

correlation time (Tc) of 1.7 nsec was

estimated for the attached spin label, at room temperature. Since changes in molecular

conformation can be observed through their

effect on the rotational motion of the spin label, a temperature-induced tural study was done by measuring T c as a function of temperature

struc-

(from 5 ° to 75°).

The results are presented in Figure 4 in the form of an Arrhenius plot of -Log T c against the inverse of the absolute temperature. is apparent at 50 ° and corresponds

A discontinuity

to the spin denaturation

in the slope

temperature

(Tsp).

This process was found to be reversible when the temperature was decreased.

The

data presented here are similar to those previously reported for other spin-labeled tRNA molecules 9-II and have been interpreted by a model in which the motion of the spin label is governed by different processes at high and low temperatures.

The

¢ Apparently, Yang and Soll 2° have recently obtained a high yield of modification of s2U * in tRNA Glu using a bromomethyl fluorescent probe. However, they have used experimental conditions (water ! dimethyl sulfoxide i:i0 as solvent) in which the macromolecule assumed a denaturate state.

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Vol. 55, No.4,1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

-/ Y

f

I

Fig. 3. EPR spectra of (a) the spin labeled tRNA Glu in the native form at pH 7.5 in a 0.02 M Tris-HCl buffer containing i0 mM MgCI2; (b) the spinlabeled nucleotide eluted from the DEAE-cellulose column.

motion of the label below T has an activation energy of 4.5 ¥ 0.2 kcal/mole sp while for temperatures above T , a value of 5.7 ~ 0.4 kcal/mole was calculated. sp

DISCUSSION

According to the recent x-ray work of Kim et al. s on tRNA Phe, the anticodon is located at one end of the "L" shaped tRNA molecule and is not buried inside the macromolecule.

This seems to be a common feature among the-tRNA's

studied so far (for a review see ref. 2).

The value of T c = 1.7 nsec and the

shape of the EPR spectrum obtained for the spin-labeled tRNA Glu in solution seem to agree with such an orientation of the anticodon loop. The results of the temperature variation study differ somewhat from those reported previously 9 under almost identical conditions of ionic strength and pH, for Val-tRNA Val spin-labeled on the amino acid.

However, the similarity of

the values found for the activation energy below T suggests that in that tempesp rature range, the dominant motion is the rotation of the spin label itself and not

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 4, 1973

T(°C) 10 I

8.50

20 I

30

40

50

60

70

80

|

I

i

I

I

I

Glu t-RNA

8.75

9.O o

50 ° @ i

9.2,5

9.50

9.75 3.6

I

I

]

I

I

I

I

3.5

3.4

3.3

3.2

3.1

3.0

2.9

___1 x 1 0 3 ( O K T

2.8

"1 )

Fig. 4. Dependence of the spin label correlation time upon absolute temperature for spin-labeled tRNA Glu in a 0.02 M Tris-HCl buffer (pH 7.5) containing i0 m M M g C I 2 .

a gradual tRNA conformational change. tion energy above T

But the fact that the T and the activasp observed for the tRNA Glu spin-labeled at the anticodon are

sp respectively 20 ° and 6 kcal/mole below the values reported for Val-tRNA Val, spinlabeled at the CCA end of the tRNA molecule, ring two different phenomena.

seems to indicate that we are monito-

Of course, the fact that the melting process might

be different for each individual tRNA, as recently put forward on theorical grounds by DeLisi 22 and the fact that there might be a conformational difference between charged and uncharged tRNA, although the question is still open 23, lwust also be taken into consideration. Nevertheless, one can always speculate on the nature of the transition observed in t~NA Glu.

Thermodynamic studies on the thermal denaturation of

tRNA22,2g, 2B have whown that the disruption of the native tRNA to a random coil structure is not an all-or-none process, but rather a step - wise melting. Hence, we can suppose that for spin-labeled t ~ A Glu, the transition might be associated with a localized dissociation of the anticodon stem leading to an intermediate state between the native and the random coil structure 2S.

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Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

therefore be impossible for the spin label to monitor the subsequent dissolution of the remaining ordered regions of the partially melted macromolecule;

thus

explaining the transition below the expected value of 60 ° to 70 ° associated with the process of denaturation

in general 22.

It must also be mentioned that the observed melting temperature might be the result of a local conformational

change brought about into the anticodon re-

gion by the introduction of the spin label.

However,

it is conceivable

that the

presumed perturbance will affect the base stacking of the anticodon loop rather than the base pairing of the anticodon stem.

Since it appears24, 25 that conside-

rable base stacking still occurs in the completely melted tRNA, this small disturbance of the base stacking should not alter significantly

the melting charac-

teristics of the anticodon stem which are associated mainly with a breakdown of base pairs24, 26 It is clear, however, cally spin-labeled

that the EPR spectra of a tRNA molecule specifi-

can provide important information pertaining to the local

organization of a specific region of the tRNA as well as secondary and tertiary structures of the whole molecule.

ACKNOWLEDGMENT S

This work was supported by Grant no. A-6413 of the National Research Council of Canada post-doctoral

(NRCC).

A.R.M.

and M.C. would like to thank the NRCC for a

fellowship and a studentship

respectively.

REFERENCES

1.

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Haar, F., Maelicke,

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Hoffman, B.M., Schofield, P

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McIntosh, A.R., Caron, M. and Dugas, H., unpublished results.

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DeLisi, C., Biopolymers 12___,1713 (1973).

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