Modifications of nucleosides and nucleotides

2~2

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95562

MODIFICATIONS OF NUCLEOSIDES AND NUCLEOTIDES VI. T H E REACTION OF GIRARD-P REAGENT W I T H T R A N S F E R RNA*

K I Y O M I KIKUGAV~A, A K I l l A MUTO, H 1 K O Y A HAYATSU**, K I N - I C H I R O M I U R A AND TYUNOSIN UKITA

Faculty o/ Pharmaceutical Sciences, University o/ Tokyo, Tohyo and Faculty o/ Science, Nagoya University, Nagoya (.Japan) (l(eceived J u n e 17th, 1966)

SUMMARY

I. Girard-P reagent (acetohydrazide pyridinium chloride) specifically converted the cytidine residues of yeast transfer RNA (tRNA) into 4-deamino-4-acetohydrazidopyridinium cytidine residues without altering any other bases. 2. The tRNA modified with Girard-P reagent was completely hydrolysed bv alkali or snake-venom phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.L4.I). A quantitative estimation of the modified cytidine residues in the tRNA was achieved by hydrolysis with the phosphodiesterase and subsequent separation of the nucleotides by ion-exchange column chromatography and estimation of the base composition. 3. The results of column chromatography and sedimentation analysis of the tRNA modified with Girard-P reagent indicated that no splitting of the internucleotide bonds of the tRNA occurred during the reaction. 4. The behavior of the modified tRNA in ion-exchange column chromatography indicated that the tRNA had less negative net charge than the native tRNA after the modification reaction. 5. The temperature-absorbance relationship plotted for the modified tRNA indicated that the secondary structure in the tRNA remarkably decreased after the modification. 6. The effect of the Girard-P modification on the aminoacylation of tRNA for tyrosine, alanine and valine was investigated.

INTRODUCTION

In the previous paper of this series, it has been reported that Girard-P reagent (acetohydrazide pyridinium chloride), similarly to semicarbazide 2-4, replaces the Abbreviations: t R N A , transfer ribonucleic acid; G r - t R N A , t R N A modified with Girard-P reagent; GrC, 4 deamino 4-acetohydrazidopyridinium eytidine. " A preliminary r e p o r t of a p a r t of this work has been communicatecP. ** Present address: I n s t i t u t e for E n z y m e Research, 17niversity of Wisconsin, Madison, Wisc., b.S.A.

Biochin~. Biophys. Aclu, 134 (t967) 232 242

REACTION OF GIRARD-P WITH NUCLEIC ACIDS

233

C-4 amino group of cytidine without affecting adenosine, guanosine or uridine 5. This time the effect of Girard-P reagent on nucleic acid was investigated and it was found that the reaction is also specific for cytidine groups in RNA. This paper deals with the base analysis, physicochemical and biochemical properties of the modified transfer RNA (tRNA).

MATERIALS AND METHODS

Yeast tRNA Crude Torula yeast tRNA was purified according to the method of MONIER6.

Enzymes The purified snake-venom phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) of Mamushi (Agkistrondon halys blomho//i BOIE) was a gift from Professor T. SUZUKI .aND Dr. S. IWANAGAof the Institute for Protein Research, University of Osaka, to whom the authors are gratefully indebted. Bovine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing), EC 2.7.7.16) (ribonuclease A) was purchased from Worthington Biochemicals Co.

Reaction o~ Girard-P reagent with tRNA A mixture of 5o mg of tRNA (sodium salt) and 2.5 ml of 2.o M Girard-P reagent solution, which contained io g Girard-P reagent and 2 M acetic acid in a total volume of 25 ml (pH 4.2), was incubated at 37 ° for 86 h. The ratio in weight of tRNA to the reagent was 1:2o. The mixture was adjusted to pH 8 with 2 M NaOH and followed by successive dialysis against o.i M NaC1 solution for 24 h and water for 48 h. On lyophilization, 49 mg of Gr-tRNA was obtained. The ultracentrifuge patterns given in Fig. 5 indicate that the modified tRNA was homogeneous.

Degradation o~ Gr-tRNA with alkali and separation o~ the nucleoside 2'(3')-phosphates A mixture of I mg of Gr-tRNA and I ml of 0.3 M KOH was kept at 37 ° for 18 h. The mixture was then carefully neutralized with 5.8 M HC10, to pH 6- 7 and cooled in an ice bath. The precipitated KC104 was removed by eentrifugation and the supernatant was analysed according to KATZ AND COMB'Smethod 7. Thus, a i-ml aliquot was withdrawn from the supernatant and added to I ml of o.I M HC1 and the mixture was applied to a Dowex 5o-X 4 (H +) column (1. 4 cm ×3-5 em). Uridine (and ~ uridine) 2'(3')-phosphate was eluted with 0.05 M HC1 in the first 15 ml effluent. On subsequent elution with water of the column, guanosine 2'(3')-phosphate was eluted in the first 8 ml effluent. The second 4 ° ml effluent of this elution was submitted to a further separation using a Dowex I-X2 (formate) column (I cm × I em). Elution of the Dowex I column with 12 ml of 0.05 M formic acid gave the fraction containing cytidine 2'(3')-phosphate and successive elution with io ml of o.4 M formic acid gave adenosine 2'(3')-phosphate. Biochim. Biophys. Acta, i34 (I967) 232 242

K. KIKUGAWAet al.

234

After exhaustive elution of the Dowex 50 column with water, the column was further eluted with I M HC1, the eluate contained decomposition products of the modified nucleotide.

Degradation o~ Gr-tRNA with snake-venom phosphodiesterase and separation o~ the nucleoside 5'-phosphates A mixture of 2 mg of Gr-tRNA, 2 rnl of o.oi M CaCI2, 2 ml of 0. 4 M Tris-HC1 (pH 8.9) and the enzyme solution (total absorbance o.o8 at 280 m/,) was incubated at 37 ° for 24 h. The reaction was stopped by addition of 4 ml of o.I M HC1 and the nucleoside 5'-phosphates produced were separated similarly by the method of column chromatography used for the alkaline hydroly~ate of the modified tRNA. Different from the case of the alkaline hydrolysate, the modified nucleotide was not degraded during the treatment of Gr-tRNA with the enzyme. It was adsorbed on the Dowex 50 column and fractionated by use of 0.5 M NH4OH after elution with 0.05 M HC1 and subsequent exhaustive elution with water of the column. The phosphomonoesterase activity in the enzyme preparation was considered to be negligible because only 4.7 °o of A-5'-P was converted to adenosine after incubation of A-5'-P with this enzyme preparation under the above conditions.

Speetrophotometric estimation o~ mtcleolides obtained by degradation o/ the modified t RNA The nucleotide fractions obtained by column chromatography of the alkaline hydrolysate and enzymic digest of the modified tRNA were adjusted to pH 2.o except in the case of GrC-5'-P and submitted to spectrophotometric estimation. The following absorption coefficeints (e~i) at given wavelength were used: U-2' (3')-P, U-5'-P (mixed with the corresponding ~ U phosphates), 9.6" IO-a at 26o m~; C-2'(3')-P, C-5'-P, I2.5-IO -a at 280 mu; A-2'(3')-P, A-5'-P, ~4.6"IO a at 26o m],; G-2'(3')-P, G-5'-P, I I . 8 . IO a at 26o m/,. The estimation of GrC-5'-P was performed at pH 12.o using eM of 18.9" IO-3 at 3o5 mu. Unless otherwise mentioned, the ratio of absorbances at 28o m], and 26o m,u of each fraction was in good agreement with the theoretical value. RESULTS

AND

DISCUSSION

Reaction o~ Girard-P reagent with yeast tRNA and quantitative analysis o~ tho~nucleotide composition o~ the modi/ied tRNA Torula yeast tRNA was incubated with 2 M Girard-P reagent solution at pH 4.2 and 37 ° for 86 h. In order to analyse the nucleoside groups in the modified tRNA, it was desirable to hydrolyse the tRNA completely to mononucleotide without altering the modified cytidine nucleotide. Alkaline hydrolysis with 0.3 M KOH at 37 ° for 18 h, usually used in the hydrolysis of ribonucleic acid, was found to cause a degradation of the modified cytidylic acid residues and cannot be used to determine GrC groups in the modified tRNA. This method, however, can be used to determine the extent of the modification of cytosine moieties by measurement of the C/A or C/G ratios before and after the modification reaction. Biochim. Biophys..4cta, 134 (1967) 2 3 2 242

REACTION OF G I R A R D - P WITH NUCLEIC ACIDS

235

In Fig. I a typical separation pattern is given of the alkaline hydrolysate of the modified t R N A by KATZ AND COmB's method 7. It shows that degradation products of the modified cytidine 2'(3')-phosphate groups are fractionated with I M HC1 after exhaustive elution of the Dowex 5o column with water. Each of the fractionated nucleoside 2'(3')-phosphates was estimated b y spectrophotometry. As is shown in Table I, the ratio of the quantities of A-2'(3')-P and G-2'(3')-P was practically equal to that obtained for native tRNA, while the ratio of C-2'(3')-P to A-2' (3')-P decreased with increased reaction time. The ratio of U-2' (3')-P (including

~//

o.e

Degradation product of 1-f a (GrCp)

~

(HCO0-)

08

(H*)

/~/ 0.~

o° 0 . 4 eJ

Dowex 1

Dowex 50

o.8i

°0.4

Io~l Gp

0.2

0.2 to dowex

1

- .~-~.

,

40

60

0I

,

8O

f

20

16o

t

t

O.05M 0.4M HCOOH HCOOH

1MHCI

O,05M H20 HCI Eff luent(m

40

t

I)

Fig. i. S e p a r a t i o n of nucleoside 2 ' ( 3 ' ) - p h o s p h a t e s in t h e alkaline h y d r o l y s a t e of G r - t R N A . E x p e r i m e n t a l m e t h o d s are g i v e n in t h e t e x t . T h e flow r a t e was 0.5 m l / m i n . T h e s h a d o w e d a r e a s r e p r e s e n t t h e fractions c o n t a i n i n g t h e nucleoside 2'(3")-phosphate. Up, Gp, Cp, Ap a n d GrCp c o r r e s p o n d to uridine, g u a n o s i n e , cytidine, a d e n o s i n e a n d 4 - d e a m i n o - 4 - a c e t o h y d r a z i d o p y r i d i n i u m c y t i d i n e 2 ' ( 3 ' ) - p h o s p h a t e s , respectively.

TABLE I THE BY

MODIFICATION ALKALINE

EXTENT

AS ESTIMATED

BY

DIRECT

SPECTROPHOTOMETRY

AND

HYDROLYSIS

C o n d i t i o n s for t h e m o d i f i c a t i o n r e a c t i o n are described in t h e legend of Fig. 3. R e a c t i o n p e r c e n t ages were c a l c u l a t e d f r o m t h e a m o u n t of t h e i n t a c t c y t i d y l y l residues. (Aal 0(pH 13) A310(pH 7))/ A2e0(pH 7) v a l u e is t h e increase in a b s o r b a n c e a t 31o m # at p H 13 relative to a b s o r b a n c e at 260 m/~ at p H 7 of t h e modified t R N A .

IRNA's

Native G r - t R N A modified for 8. 5 h G r - t R N A modified for 35 h G r - t R N A modified for 95 h

Base ratios as determined by alkaline hydrolysis Ap/Gp

Cp/Ap

Up/Ap

0.68 0,64 0,65 0,63

1.14 1.o 5 0.73 0.35

1.38 1.43 1.66 1.79

Reaction ( °,o)

o 8 36 69

A.'~1o(pH 13)--A31o(pH 7)

A26o(pH 7)

0.063 o.129 0.269

~uU phosphates) to A-2' (3')-P obtained from the modified tRNA, however, was larger than that obtained for the native tRNA. The increase in the amount of uridine phosphates appears not to be due to the actual increase of uridylic acid, because as was indicated in the previous paper 5, when GrC-2'(3')-P was submitted to similar alkaBiochim. Biophys. Acta, 134 (1967) 232-242

236

K. KIKUGAWA el al.

line t r e a t m e n t as used for the h y d r o l y s i s of the modified t R N A , the reaction solution c o n t a i n e d two u n k n o w n d e g r a d a t i o n products, one of which was f r a c t i o n a t e d b y D o w e x 5o c h r o m a t o g r a p h y in U - 2 ' ( 3 ' ) - P fraction, b u t m o v e d differently in p a p e r electrophoresis. Moreover, at p H 2 this p r o d u c t showed a sinfilar A2so/A2G o ratio to t h a t of U - e ' ( 3 ' ) - P . The best m e t h o d found so far for complete d e c o m p o s i t i o n of the modified t R N A to mononucleotides w i t h o u t a n y d e g r a d a t i o n of the base groups was digestion with s n a k e - v e n o m p h o s p h o d i e s t e r a s e s. This enzyme c o m p l e t e l y d e c o m p o s e d the m o d ified t R N A into nucleoside 5 ' - p h o s p h a t e s , A - 5 ' - P , G - 5 ' - P , U-5'-P, C - 5 ' - P a n d G r C - 5 ' - P , a n d a n y further d e c o m p o s i t i o n of G r C - 5 ' - P b y the enzyme was not observed. A f t e r the digestion, these nucleotides were effectively s e p a r a t e d in a similar w a y b y t h e m e t h o d of column c h r o m a t o g r a p h y described above. In Fig. 2, the sepa r a t i o n of the nucteoside 5 ' - p h o s p h a t e s b y means of ion-exchange columns is given. The procedure of the s e p a r a t i o n was similar to t h a t used for the alkaline h y d r o l y s a t e of the modified t R N A . Dowex 1 (HCO0-)

Dowex 50 (H % 0.4 7"7]

///

04

0.4

111

//z

,//A

GrCp

Upl e p

o o,a .YA •

¢/A

Submitted to dowex 1 • ,.

40

t O.05M HCI

®

o ::).2 ~

/A

H20

,~

,

60

g

~0.2

~///'/I

•.---[///2-m.

l

80

.

100

0.5 M NH40H Effluent (rnl)

O0

20

1 O.05M

1 O.4M

40

HCOOH HCOOH

Fig. 2. Separation of nucleoside 5'-phosphates in the snake-venmn phosphodiesterase digest of (;r-tRNA. Experimental methods are given in the text. The flow rate was o. 5 ml/min. The shadowed areas represent the fractions containing the nucleoside 5'-phosphate. Up, Gp, Cp, Ap and GrCp correspond to uridine, guanosine, cytidine, adenosine and 4-deamino-4-acetohydrazido pyridinium cytidine 5'-phosphates, respectively. GrCp was estimated at 305 m# in alkaline solution at p H ] 2.

Fig. z indicates t h a t G r C - 5 ' - P is a b s o r b e d on the D o w e x 5 ° column a n d is e l u t e d with 0.5 M NH4OH after e x h a u s t i v e washing of the column w i t h water. E a c h nucleoside 5 ' - p h o s p h a t e thus s e p a r a t e d was e s t i m a t e d b y s p e c t r o p h o t o m e t r y . The ratio of GrC p h o s p h a t e s plus cytidine p h o s p h a t e s to adenosine p h o s p h a t e s (I.19) o b t a i n e d from the digest of the modified t R N A , was in good accord with t h a t of cytidine p h o s p h a t e s to adenosine p h o s p h a t e s (I.2o) o b t a i n e d from the n a t i v e t R N A a n d this result shows t h a t all the c y t i d i n e groups in t h e n a t i v e t R N A , which r e a c t e d with G i r a r d - P reagent, were c o n v e r t e d to GrC a n d t h a t this procedure is a v a i l a b l e for the e s t i m a t i o n of the e x t e n t of the modification. Biochim. kliophys. Acta, 1.34 (t967) z3z z4z

237

REACTION OF GIRARD-P WITH NUCLEIC ACIDS

In Table I I the cytidine phosphates to adenosine phosphates ratio obtained b y alkaline hydrolysis and enzymic digestion of the modified tRNA, which was prepared by reaction of t R N A with Girard-P reagent at p H 4.2 and 37 ° for 86 h, are compared with those found for the native tRNA, and the ratio of the quantities of cytidine phosphates in both the t R N A ' s is also given. The table indicates that the results obtained from the alkaline hydrolysis and the enzymic digestion are in agreement and that in the modified tRNA, approx. 7o-75 % of cytidine residues were converted to GrC groups.

TABLE I[ COMPARISON

OF THE

AS D E T E R M I N E D

INTACT CYTIDYLYL

BY ALKALINE

AND

GROUPS

ENZYMIC

IN MODIFIED

AND NATIVE

tI{NA's

HYDROLYSES

Conditions for alkaline hydrolysis and separation of the nucleoside 2 ' ( 3 ' ) - p h o s p h a t e s are given in the legend of Fig. i, and t h o s e for enzymic; hydrolysis and s e p a r a t i o n of the nucleoside 5'p h o s p h a t e s are given in the legend of Fig. 2. See also EXPERIMENTAL. G r - t R N A which was prepared b y the reaction of t R N A and 2 M G i r a r d - P (pH 4.2) at 37 ° for 86 h was used.

Hydrolysis with

Cp/A p

Cp residues in Gr-I R N A Cp residues in t R N A

o. 3 M K O H

tRNA Gr-tRNA

1.26 o.32

o.254

Snake-venom phosphodiesterase

tlRNA Gr-tRNA

1.2o o.37

o.3o8

Reaction rate The rate of the reaction between Girard-P reagent and t R N A was determined by following the extent of the modification of the cytidine residues in the tRNA. Thus t R N A was incubated with 2 M Girard-P reagent at pH 4.2 and 37 ° and aliquots were withdrawn at different time intervals and the modified tRNA's isolated. After alkaline hydrolysis of the modified tRNA's, the ratios of cytidine 2' (3')-phosphate to adenosine 2' (3')-phosphate in the hydrolysates were determined and some of the values obtained are presented in Table I. In Fig. 3, the reaction rate was plotted against time and it shows that after 5o h of the reaction approx. 5o °o of the total cytidine groups in the t R N A are modified by the reagent.

Hydrolysis o/ Gr-tRNA with bovine pancreatic ribonuclease The modified t R N A which was prepared by incubation of t R N A with 2 M Girard-P reagent at p H 4.2 and 37 ° for 86 h was limitedly digested with bovine pancreatic ribonuclease (ribonuclease-A) at 28~o.5 ° for 30 min and at a substrate/enzyme ratio of 3ooo:1 (w/w). The nucleoside 3'-phosphates, GrC-3'-P, C-3'-P and U-3'-P (including ~r/U-3'-P), produced were separated and determined. The molar ratio of GrC-3'-P and U-3'-P obtained was I :30. In Fig. 4, the separation and determination of the nucleotides are given. Biochim. Biophys. Acta, 134 (1967) 232-242

238

K. KIKAGAWA 6t

al.

10 100

oO4

8c

O.6

6c

E 40

A pC - 3'- P

_ ,_

0.2 GFC-3'-P

~5 o

~0 2 0 x Ld

,

o

20

t Ha0

0

2'0

4'0

dO

8'0

40

I

60

°)

O.O01M

160

He'

80

lOO

12o

O.OlM

0.025

14o

16o

\ .OO3M

HCi

HCi

Time(h)

M

HCi

Effluent (ml)

Fig. 3. R e a c t i o n r a t e in t h e m o d i f i c a t i o n of t R N A w i t h Girard-I ) reagent. A solution of IOO m g of t N N A in 3 ml of 2.o M Girard P r e a g e n t solution (pH 4.2) was k e p t at 37". A o.6-ml a l i q u o t was w i t h d r a w n at different t i m e i n t e r w d s . T h e modified t N N A w a s isolated a n d h y d r o l y s e d w i t h o. 3 M K O H . T h e nucleoside 2 ' ( 3 ' ) - p h o s p h a t e s were s e p a r a t e d a n d d e t e r m i n e d b y s p c c t r o p h o t o m e t r y as described in t h e t e x t . Fig. 4. S e p a r a t i o n of nueleoside 3 ' - p h o s p h a t e s p r o d u c e d b y limited digestion of ( ; r - l i a N A with bovine p a n c r e a t i c ribonuclease. A solution of 15 m g of G r - t R N A , w h i c h was prepared b y reaction of t R N \ with 2 M G i r a r d - P r e a g e n t at p H 4.-' a n d 37 ° for 86 h (approx. 7o % of t h e total cytidine residues were modified), in 3 ml of ribonuclease solution (contained 5 # g of t h e e n z y m e in o.o 5 M Tris-HC1 buffer (pH7.5)) was i n c u b a t e d at 2 8 ± o . 5 ° for 3 ° rain. T h e m i x t u r e was k e p t at p H z a n d r o o m t e m p e r a t u r e for 3 ° rain, a n d s u b s e q u e n t l y centrifuged. T h e s u p e r n a t a n t was n e u t r a l i s e d w i t h N H 4 O H , applied o n t o a D o w e x I-X2 (C1) c o l u m n (1 c m :. 2. 5 cm) a n d t h e colm n n eluted as s h o w n in t h e figure. T h e nucleotides s e p a r a t e d were d e t e r m i n e d b y s p e c t r o p h o t o m e t r v . T h e s h a d o w e d area indicates U - 3 ' - P eluted w h i c h was a n a l y s e d as follows. To t h e conlb i n e d fractions from IOO to 18o ml, w h i c h c o n t a i n e d several oligonucleotides besides [ T 3'-I ), was a d d e d 2 . 1 m l of i M HC1 a n d it was applied onto a D o w e x 5 ° (H +) c o h m m (i c m . 3 . 5 c m ) a n d t h e c o l u m n w a s h e d with 1o ml of o.o 5 M HC1. The t o t a l e f f l u e n t x~as n e u t r a l i z e d w i t h B a ( O H ) 2 , c o n c e n t r a t e d a n d t h e c a t i o n s r e m o v e d b y D o w e x 5 ° (Hq) resin. An a l i q u o t of t h e cation-free s o l u t i o n (total a b s o r b a n c c 6.o 4 at 26o raft) w a s s u b m i t t e d to p a p e r c h r o m a t o g r a p h y (solvent: iso p r o p a n o l 6 M HC1 w a t e r (68 : 33.5 :1.5, v/v)). T h e s p o t of [" 3 ' - P (Rt," o.83) was e x t r a c t e d w i t h O.Ol M HC1 a n d e s t i m a t e d 1)y s p e c t r o p h o t o m e t r y .

Tile results idue

and

readily

indicate

the 5'-position

that

split by tile enzyme

becomes

remarkably

replaced

by (;rC groups.

the internucleotide

of t i l e n e i g h b o r i n g than

resistant

the corresponding

to the enzyme

linkage between

nucleoside, action

tile C-3'-P

w h i c h is k n o w n

linkage when

of the U-3'-P the cytidine

res-

to be more residue,

groups

are

Physical properties o~ G r - t R N A i n o r d e r t o c h e c k if t i l e i n t e r n u c l e o t i d e modification and

reaction,

the Gr-tRNA

l i n k a g e of t R N A

was submitted

was split during the

to ultracentrifugation

analysis

to ion-exchange chromatography on a DEAE-cellulose column. The ultracentrifuge patterns indicated that the modified tRNA was homoge-

neous native

( F i g . 5). T h e S v e d b e r g

values obtained

tRNA and 4.o0 for Gr-tRNA. In Fig. 6 the chromatographic

patterns

Biochim. Biophys. ,4cta, I34 ( 1 9 0 7 ) 2 3 2 242

from these patterns

were 4.o5 for the

of t h e n a t i v e

and Gr-tRNA

tRNA

on

18o

REACTION OF GIRARD-P WITH NUCLEIC ACIDS

230

Fig. 5. Sedimentation patterns of native t R N A (A) and Gr-tRNA which was prepared by reaction of 2 M ¢;irard-P reagent with tI
a DEAE-cellulose column are given, which show that both the tRNA's were eluted, e a c h g i v i n g a s i n g l e p e a k . T h e p e a k of t h e n a t i v e t R N A a p p e a r e d a f t e r e l u t i o n w i t h o.5 M NaC1, w h i l e t h a t of t h e m o d i f i e d t R N A a f t e r o.4 M NaC1 e l u t i o n , i n d i c a t i n g t h a t t h e l a t t e r t R N A w a s less n e g a t i v e l y c h a r g e d . T h e s e r e s u l t s c l e a r l y i n d i c a t e t h a t n o g r o s s s p l i t t i n g of t h e i n t e r n u c l e o t i d e l i n k a g e of t h e t R N A o c c u r r e d d u r i n g t h e modification reaction.

f JJJ

lo

o

s

-,'>--

/

50

o,s~ Z

././ o

tO

i

- - , - / - - - - - +1- 0" -0"

\ 150

200

E l f u e n t (ml)

Fig. 6. Chromatographic patterns on a DEAE-cellulose column of the native t R N A and Gr~ tRNA. Each native t R N A (5 mg) and Gr-tRNA (I 5 mg) which was prepared by reaction of 2 M Girard-P reagent with t R N A at pH 4.2 and 37 ° for 41 h was loaded on DEAE-cellulose columns (1.8 cnl >(6.o cm) equilibrated with o.1 M Tris-HC1 (pH 7.5) and chronlatographed by a linear gradient elution using a nlixture of o.i M Tris HC1 (pH 7.5) and o to i M NaC1. O - - - O , native tRNA; O - - O , Gr-tRNA.

T h e u l t r a v i o l e t a b s o r p t i o n s p e c t r a of t h e n a t i v e t R N A a n d t h e m o d i f i e d t R N A , i n w h i c h 7 ° 0//o of t h e t o t a l c y t i d i n e g r o u p s w e r e m o d i f i e d b y G i r a r d - P r e a g e n t , w e r e r e c o r d e d i n w a t e r a n d i n o.o5 M N a O H ( p H I 3 ) a n d a r e g i v e n i n Fig. 7. T h e spectra_ w e r e s t a b l e i n t h e a l k a l i n e s o l u t i o n for IO m i n a f t e r p H a d j u s t m e n t . Biochim. Biophys. Acta, 134 (1967) 232-24z

K. KIKAGAWAel al.

240

As was reported in the previous paper s, cytidine modified with Girard-P reagent shows a distinct ultraviolet absorption maximum (
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220

260

300

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Fig. 7. Ultraviolet a b s o r p t i o n s p e c t r a of the native t R N A and G r - t R N A . The spectra of n a t i v e t R N A () and G r - t R N A which was p r e p a r e d b y reaction of t R N A with 2 M Girard-P reagent for 95 h ( - ) were t a k e n in w a t e r (a) and in o.o 5 M N a O H (b).

The effect of temperature on the ultraviolet absorption at 26o m/, of the modified tRNA dissolved in o.I M phosphate buffer (pH 7.o) was investigated and the results are given in Fig. 8. The hvperchromicitv_ . of the modified t R N A was approx. 8 O,.o upon heating from 2o ° to 9 o°. This value was remarkably smaller than that of the native tRNA, approx. 26 %. The Tm value of the modified tRNA, 46°, was lower than that of the native tRNA, 54 °. The results indicate that the secondary structure of tRNA has been largely decreased by treatment with Girard-P reagent and the decrease seems to be more marked than that caused by the modification of tRNA with semicarbazide. Thus the modified tRNA in which 88. 5 o~ of cytidine groups were converted to 4-deamino-4-semicarbazidocytidine groups showed a hyperchromicity of approx. 2o ~Io and Tm of 4 6° (ref. 4).

Effect o~ the Girard-P modification on the aminoacylalion o~ t R N A The amino acid-accepting activity of tRNA was assayed using yeast aminoacyl l~io~kim, l~iopky.~..4cta, 134 (I967) 232-2.42

R E A C T I O N OF G I R A R D - P

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W I T H N U C L E I C ACIDS

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F i g . 8. R e l a t i v e a b s o r b a n c e at 26o mff of n a t i v e t R N A and G r - t R N A as a f u n c t i o n of temperature. The curves indicate t h e plot for n a t i v e t R N A ( Q - - - 0 ) and for the t R N A treated w i t h 2 M Girard-P reagent for 95 h ( © O ) . The absorbance was measured in o . i M p h o s p h a t e buffer ( p H 7.o). Fig. 9. Effect of Girard-P m o d i f i c a t i o n on t h e a m i n o acid-accepting a c t i v i t y of T o r u l a - y e a s t t R N A . The relative a c t i v i t y of t h e i n t a c t t R N A was p l o t t e d against t h e e x t e n t of the m o d i f i c a t i o n . Valine, O ; tyrosine, O; alanine, A .

RNA synthetase as described in a previous paper 9, after yeast t R N A was treated with Girard-P reagent for different time intervals as described above. The rates of inactivation of aminoacylation of tRNA for tyrosine, alanine and valine after treatment with Girard-P reagent were quite similar to each other as shown in Fig. 9, and the inactivation was a one-hit event. This is different from the case of semicarbazide modification 9, in which t R N A loses its amino acid-accepting ability at different rates for each kind of amino acid. Girard-P reagent might more effectively modify the common cytidylic acid residues which are essential for accepting activity for every kind of amino acid. Another possible reason for the common inactivation might be a change in the electrostatic charge of the tRNA molecule owing to the modification by this reagent.

Concluding remarks The results reported in this paper show that cytosine groups of RNA can be specifically modified by Girard-P reagent without decomposition of the nucleic acid. The phosphate bonds of the GrC 3'-phosphate residues in the modified tRNA were found to be highly resistant to hydrolysis by bovine pancreatic ribonuclease as compared with those of the cytidine J-phosphate residues in the original tRNA. This change in the susceptibility of tRNA to the enzymic reaction would be of obvious advantage fol the sequence analysis of nucleic acids. The effect of the Girard-P modification on the aminoacylation of tRNA was different from that of the semicarbazide modification 9. Thus, aminoacylations of tRNA for tyrosine, alanine and valine were inactivated quite similarly after treatment of t R N A with Girard-P reagent. Biochim. Biophys. ,4cla, 134 (1967) 2 3 2 - 2 4 2

K. K I K U G A W A e[

242

al.

REVERENCES

[ T. [:KYrA, lq. I(H;U(;..XW5 ANI) H. H..\YATSU, 6tk l~tern. ('ongr. l¢i¢)cke,t. Nezt, Yor/¢, 2964, :hi)st. ~, S e c r e t a r i a t 6 t h I n t e r n . ( ' o n g r . l~iochem., \ V a s h i n g t o n 1). ('., [LS..X., p. 91. 2 H. HAY.XTSU : \ N I ) T . [TKI'r.\, Hioc]W,~. lHophys. Nes. Com,tzt,., I t ( 1 9 6 4 ) t 9 8 . :3 H . HA'v.',l'st,, 1"4. "I'.xI-:]~:ISHI .",NI)T. [TKITA, 13iochi,I. l~i~)ph3's .qc/a, ~23 ( 1 0 0 0 ) 4454 H. H.xv.\'I'su .\NI> IC ['I
I¢i~,cki,I1. f~i(@ky.<. .tcla,

13t (i()~)7) "-32 242