the primary structure of aspartate transfer ribonucleic acid from brewers yeast: ii. partial...

21o BIOCHIMICA ET BIOPHYSICA ACTA

BB#, 97080

THE PRIMARY STRUCTURE OF ASPARTATE TRANSFER

RIBONUCLEIC ACID FROM BREWERS YEAST

II. PARTIAL DIGESTIONS WITH PANCREATIC RIBONUCLEASE AND

T 1 RIBONUCLEASE AND DERIVATION OF COMPLETE SEQUENCE

J. GANGLOFF, G. KEITH, J. P. EBEL AND G. DIRHEIMER Laboratoires de Chimie biologique de la Facultd des Sciences et de la Facultd de Pharmacie, Strasbourg (France)

(Received July i3th, 1971)

SUMMARY

Specific cleavage of tRNA A~p at the anticodon site with T~ ribonuclease enables the isolation of two halves of the molecule. Additional partial hydrolyses with pancreatic and T~ ribonucleases yield large oligonucleotide fragments for characterisa- tion. A carbodiimide modification of tRNA Asp followed by pancreatic ribonuclease digestion was necessary for the determination of the primary structure of Half- fragment I. The information provided by these analyses and those described in the preceding paper have permitted the derivation of the total sequence of tRNA A~p.

INTRODUCTION

The results obtained by total pancreatic ribonuclease and T 1 ribonuclease digestions of tRNA Asp from brewer's yeast reported in the preceding paper 1 did not give enough overlaps for the derivation of the primary structure of this tRNA. I t was therefore necessary to carry out partial digestions. This paper reports the detailed conditions of controlled cleavage of tRNA Asp with T 1 and pancreatic ribonuclease and the methods used for the separation and analyses of the large oligonucleotide fragments produced. The nucleotide sequence is deduced from overlaps amongst the large oligonucleotides produced. Similarities between tRNA Asr and other tRNAs for which the sequences are known are discussed.

MATERIAL AND METHODS

General Methods

The source of tRNA As~ and of the various enzymes, the conditions and solvent systems used for thin-layer chromatography and high voltage electrophoresis, the methods of analysis and identification of oligonucleotides and other general techni- ques used were described previously 1.

Chromatography on D E A E-cellulose columns

Column chromatographies were carried out on DEAE-cellulose (Schleicher and Schtill, No. 7 o, about 0. 9 mequiv, per g) prepared as previously described 1.

Biochim. Biophys. Acta, 2.59 (1972) 21o--222

PRIMARY STRUCTURE OF YEAST KSPARTATE t R N A . I I 211

Before use, the cellulose was equilibrated with a 7 M urea-o.o2 M Tris-HC1 (pH 7.3) buffer and degassed in vacuo at 60 °. For packing long thin columns (usually 0. 7 cm × 25 ° cm), a fine slurry of this DEAE-cellulose suspension was poured into a column

filled wid~ the buffer. The bottom of the column was connected to a peristaltic pump establishing a flow rate of 2 ml/min. The column was then washed thoroughly with the same buffer before use. Approximately 5-Io-ml fractions were collected.

When column chromatography was performed at acidic pH, the DEAE cellulose was suspended in a 7 M urea solution, adjusted to pH 3.0 with concentrated HC1 before pouring it in the column. This solution was also utilized for washing the column before use.

Desalting Urea and salt were separated from the pooled oligonucleotide fractions as

previously described 1. The effluents from chromatography performed at acidic pH were neutralized with a I M solution of Tris before desalting.

Separation o/oligonucleotide [ragments Peaks of oligonucleotides isolated from DEAE-cellulose columns at neutral

pH were further separated, after a 5-fold dilution with water, by rechromatography on DEAE-cellulose at acidic pH (ref. 2) under the conditions described above.

Analysis o/oligonudeotides Oligonucleotides of partial digestions were degraded with pancreatic or T 1

ribonuclease and then separated by high voltage electrophoresis as described in the preceeding paper 1.

Speci/ic cleavage o] tRNA A'p into two large/ragments The incubation mixture contained tRNA Asp (5.5 mg) in 0.02 IV[ cacodylate

buffer (pH 6.5) containing 0.02 M MgC12 (1.3 ml). I t was cooled in ice and mixed with o.13 ml of a cold solution of ribonuclease T 1 (500 units/ml) in the same buffer. After IO min at o °, 2.5ml of cold water were added and the reaction was stopped by adding 4 ml of a phenol solution (phenol saturated at o ° with 0.02 cacodylate buffer (pH 6.5) containing 0.02 M MgC12). The phenol phase was separated and the phenol treatment was repeated twice with 4-ml portions of phenol. The combined phenol layers were re-extracted with 4 ml of cacodylate buffer. The aqueous layers were combined and extracted 6 times with an equal volume of ether to remove resi- dual phenol and the resulting aqueous solution was then applied on a column of DEAE-cellulose.

Partial digestion o] tRNA asp with ribonuclease T 1 in the absence o] Mg 2+ 20 mg of purified tRNA Asp in I I ml of o.i M Tris-HCl buffer (pH 7.5) were

kept at o ° for 20 min and mixed with 5000 units of ribonuclea~e 1"1 dissolved in i ml of o.i N[ Tris-HC1 buffer (pH 7.5) also cooled in ice before mixing. After 30 rain at o °, the enzyme was removed by repeated extraction of the mixture with three I2-ml portions of phenol saturated with o.i M Tris-HC1 (pH 7.5) at o °. The aqueous phase was freed of phenol and made 7 M in urea as described above.

Partial digestion o / t R N A Asp wth pancreatic ribonuclease In this experiment tRNA A~p (20 mg) was incubated with pancreatic ribonu-

clease (o.I mg) in IO ml o.i M Tris-HCl buffer containing o.oi M MgC12. Incubation

Biochim. Biophys. ,4cta, 259 (1972) 2 i o 222

212 J. GANGLOFF et al.

was conducted at o ° for 3o min, removal of pancreatic ribonuclease and subsequent t reatments were the same as described above for partial digestion with T lribonuclease.

Polyacrylamide gel electrophoresis The action of ribonuclease was followed by electrophoresis of the reaction mix-

tures on polyacrylamide gel (12 %, 7 M urea (pH 8.1)). The gels were stained with acridine orange. The optimal partial hydrolysis conditions were estimated from these separations.

Treatment o[ tRN A A~p with a watersoluble carbodiimide and hydrolysis o[ the modified t R N A with pancreatic ribonuclease

5 mg of dry, alcohol precipitated, tRNA A~p were dissolved in 4.5 ml of o.i M Tris-HC1 (pH 7.5) and treated with 500 mg of N-cyclohexyl-N'-2-ethyl(-4-morpho- linyl)carbodiimide p-toluenesulphonate (Fluka pract.) during 9omin at 65 °. The tRNA was afterwards freed of the excess of reagent by a 2-fold alcoholic preci- pitation (4 vol. alcohol, o.i M NaC1).

The carbodiimide-treated tRNA was dissolved in 2 ml of o.I M Tris-HC1 buffer (pH 7.5) and hydrolysed with 5oo/zg of pancreatic ribonuclease during 2 h

at 37 ° .

RESULTS

Specific cleavage o[ t R N A asp in two halves In preliminary experiments, conditions for degradation of tRNA to yield just

two large fragments were established by treating tRNA Asp with T 1 ribonuclease at o ° at pH 6.5 and in presence of Mg ~+ as described under MATERIAL AND METHODS and by polyacrylamide gel electrophoresis of aliquots taken out after various periods of time. After IO rain the tRNA was completely hydrolysed into two fragments.

A good separation into two peaks was obtained by chromatography on DEAE- cellulose at pH 7-3 in presence of 7 M urea (Fig. I). Peaks 1 and I I (Fig. I) thus obtained were, after desalting, exhaustively digested with ribonuclease 1"1 and pancreatic ribonuclease and analysed. As shown in Table I, the analyses of these two peaks were mutual ly exclusive and were complementary to each other. The sum of the analyses of I and I I correspond, with some exceptions, to the analysis of the original tRNA Asp indicating tha t the tRNA molecule was cleaved into two large fragments, Peak I having the pUp terminal nucleotide.

The C-C-A fragment is absent in the T 1 ribonuclease digest of Peak II . One Cp and the adenosine fragment are also absent in the pancreatic ribonuclease hydrolysate of Peak II . Furthermore, the pancreatic ribonuclease fragment G-G-A-G-Cp is not found in Peak I I but is replaced by an oligonucleotide G-G-A-Gp not obtained from the original tRNA Asp digestion. These results indicate that the terminal C-C-A oligonucleotide has been cleaved by the partial digestion because it is preceeded by a G. The same result has been obtained by DOTTING et al. 8 with tRNAI ser and tRNA nser. Our results also show that the 3' terminal sequence of tRNA mp is G-G-A-G-C-C-A.

Table I shows also that a dinucleotide G-Up is lost in the pancreatic ribonuclease digest. Gp appears in Fragment [ and an additional Up in Fragment II.

Biochim. Biophys . Acta, 259 (1972) 21o 222

PRIMARY STRUCTURE OP YEAST ASPARTATE tRNA. n 213

E c

o

o~

1

.)0 1~0 I 140 t I

150 160

0~4-

Q2,-

I 170

F r e c t i o n s of 9 ml

Fig. I. DEAE-cellulose column (0.6 cm × 230 cm) ch roma tography of the part ial ribonuclease T x digest of t R N A As" (.5-5 mg). Elut ion wi th a linear gradient (2 1 total volume) of ~qaC1 (o to o. 5 M) in 0.02 M Tris (pH 7.3)-7 M urea.

This means that the partial hydrolysis has cleaved the molecule at the level of G-Up, probably after Gp of the postulated anticodon G-U-Cp (ref. I).

The results shown in Table I reveal only one overlapping: among the oligonucleotides obtained by pancreatic ribonuclease hydrolysis of Fragment I I the only one having, at the 5' terminal end, an A-G is A-G-A-U; this overlaps the following two oligonucleotides obtained by T 1 hydrolysis of Fragment II : C-C-A-G, which is the only oligonucleotide having A-G at the 3' terminal end, and A-U-msC-G which is the only one with A-U at the 5' terminal end. A hexanucleotide C-C-A-G-A-U-mSC-Gp can therefore be deduced.

This oligonucleotide overlaps with a nonadecanucleotide: A-U-mSC-G-G-G-G - T-~u-C-A-A-U-U-C-C-C-C-Gp obtained in little amounts after exhaustive digestion of tRNA ASp by T 1 ribonuclease 1. The sequence of 23 nucleotides: C-C-A-G-A-U-mSC - G-G-G-G-T-~-C-A-A-U-U-C-C-C-C-Gp (Fragment a) can be deduced from these re- suits.

Partial enzymatic digestions o/ tRNA Asp Additional partial hydrolyses were necessary to obtain the total nucleotide

sequence of tRNA Asp. We performed a partial hydrolysis with T 1 ribonuclease and another one with pancreatic ribonuclease. The optimal conditions for these partial digestions were determined by adding increasing amounts of the enzymes to the RNA and fractionating the hydrolysates by polyacrylamide gel electrophoresis.

The conditions we selected for T 1 hydrolysis were about the same as total hydrolysis 1 as far as the pH is concerned (7.5) and the relative concentration of ribonuclease to RNA (25 ° units/rag) but instead of 4 h at 37 °, we performed the reaction for 30 rain at o °.

Biochim. Biophys. Acta, 259 (1972) 21o-222

$ T

AB

LE

I

AN

AL

YS

ES

O

F

TH

E

TW

O

LA

RG

E

FR

AG

ME

NT

S

OB

TA

INE

D

BY

S

PE

CIF

IC

CL

EA

VA

GE

O

F

tlR

NA

Asp

WIT

H

T t

RIB

ON

UC

LE

AS

E

~D

~D

"..1

h~

Tot

al h

ydro

lysi

s w

ith

T 1

rib

onuc

leas

e o[

tR

NA

A,.

a

nd

o/

Fra

gm

ents

I

an

d I

I o/

Fig

. I

To

tal

hyd

roly

sis

wit

h p

an

crea

tic

ribo

nucl

ease

o

/tR

NA

a~p

a

nd

o[

Fra

gm

ents

I

an

d I

I o[

Fig

. z

Oli

gonu

cleo

tide

F

rag

men

t tR

NA

a,v

D

i[/e

- O

ligo

nucl

eoti

de

Fra

gm

ent

tRN

A A

sp

Di/

/e

renc

e ..

..

fenc

e I

II

I II

v lo

0 t¢

1¢

C-C

-A

i --

i A

de

no

sin

e

I ...

. I

Gp

3

4 7

Cp

3

8 1

2

- i

U-C

-m~

G

~>

p I

I U

p

2 2

3 +

I

Gp

I

3- I

C

-Gp

I

2 3

~rt

p 2

1 .3

U

-Gp

i

i 2

m~

Cp

i

i A

-Gp

i

I m

lG-C

p

i x

U-C

-Gp

i

i G

-Cp

i

2 3

hU

-C-A

-Gp

i

i G

-Up

I

2 4

-- I

C

-~/-

t-U

-Gp

i

t A

-A-h

Up

I

t C

-C-A

-Gp

I

I A

-A-U

p

i I

A-U

-msC

-Gp

i

i p

Up

l

i A

-A-U

-Gp

i

i G

-A-U

p

i i

A-U

-A-G

p

i i

A-G

-Up

i

i p

U-C

-C-G

p

i i

G-G

-hU

p

i i

U-U

-~E

t-A

-A-h

U-G

p

i i

A-G

-A-U

p

i i

T-~

rt-C

-A-A

-U-U

-C-C

-C-C

-Gp

i

i G

-G-G

-Cp

i

i

G-G

-A-G

p

i +

I

A-G

-A-A

-Up

i

i G

-G-A

-G-C

p

I I

G-G

-G-G

-Tp

i

i

.'7

©

v.-

©

PRIMARY STRUCTURE OP YEAST ASPARTATE tRNA. II 215

For the partial hydrolysis with pancreatic ribonuclease we worked with a reaction mixture at pH 7.5 containing o.oi M MgCI z and with 4 ° times less ribonuclease as for total hydrolysis ~. Moreover the enzyme worked only 30 rain at o ° instead of 3 h at 37 °.

Results o/ partial hydrolysis with pancreatic ribonuclease. The elution pat tern obtained upon chromatography of a partial pancreatic ribonuclease digest of tRNA A~p on DEAE cellulose is shown in Fig. 2. The brackets show the two fractions, A and B, which were pooled for rechromatography at acidic pH. Fig. 3 shows the rechromatography of Fraction A of Figs. 2 and 4 the rechromatography of Fraction B of Fig. 2 on DEAE-cellulose at pH 3, Peaks A x, A 2, B 1, B 2 and B 3 were, after desalting, exhaustively digested with 3"1 ribonuclease and submitted to a high voltage electrophoresis.

- 4

- 3

1

40 8 0 120 160 180

Fig. 2. DEAE-ce l lu lo se co lumn (0. 7 cm × 200 cm) c h r o m a t o g r a p h y of a pa r t i a l panc rea t i c ribonuclease d iges t of t R N A A~p (20 mg) E l u t i o n w i t h a l inear g r ad i en t (2 1 t o t a l vo lume) of NaCI (o to o. 5 M) in 0.02 M Tris (pH 7.3)-7 M urea.

Fragment A x (Fig. 3) yielded I/*mole of Cp for i.c;/,moles of Gp and I / ,mole of the following oligonucleotides: C-Gp, C-A-Gp and A-A-U-Gp. The presence of A-A-U-Gp places this fragment in the half of tRNA A~p bearing the 5' terminal end (Fragment I in Table I). C-Ap must be at the 5' end of Fragment A 1 because we know from total T1 ribonuclease digestion of Fragment I (Table I) that the trinucleotide C-A-Gp is preceeded by bU. Three oligonucleotides beginning with A-G were found in the pancreatic ribonuclease digestion of the 5' terminal half of tRNAA~p: A-G-Up, A-G-A-Up and A-G-A-A-Up. The A-G sequence of C-A-Gp cannot be followed either by a U or by an A-U because these fragments do not appear in the T 1 ribonuclease digest of Fragment A 1. Therefore C-A-G is followed by A-A-Up. Finally, Cp is at the 3' end of Fragment A 1 because it derives from a partial pancreatic digestion of tRNA Asp. Consequently the pr imary sequence of Fragment A 1 is C-A-G-A-A-U-G (G z, C-G)Cp. I t was very difficult to determine the sequer.ce between brackets. The method we finally selected was a carbodiimide modification of tRNA A~p. This technique, described by GILHAM 4, was carried out according to the method of G. H.


216 j , GANGLOFF~et al.

E c

o

.¢

E c

o

°

I | I 0 10 20 30

Fraction B

- - | , I I o 140

Fraction

v

o z

4

I I 180

Fig. 3. Elut ion pa t t e rn obtained upon r ech romatography of Peak A (Fig. 2) on a DEAE-cel lulose column (0. 7 cm X 20o cm) Elut ion wi th a linear gradient (I 1 total volume) of NaC1 (o.I to 0.35 M) in 7 M urea-HCl (pH 3)-

Fig. 4. Elut ion pa t t e rn obtained upon r ech romatography of Peak B (Fig. 2} on a DEAE-cel lulose column (0. 7 cm × 20o cm) Elut ion wi th a linear gradient (2 1 total volume) of NaC1 (o to o.35 M) in 7 M urea-HC1 (pH 3)-

1.~ 0.2

0.1 ~o ~ c ~

, o

0 5~ i 100 150 F r a c t i o n No.

Fig. 5. Chromatography of pancreat ic ribonuclease digestion of tRNA A'p modified by carbodiimide. The pancreat ic ribonuclease digest diluted IO times with water, was charged on a D E A E - cellulose column (60 cm x o.6 cm) and eluted wi th a linear gradient of o-o.2 1Vf NaC1 in 0.02 M Tris-HC1 buffer (pH 7.5) 7 M urea. Total volume of the gradient I 1.


PRIMARY STRUCTURE OF YEAST ASPARTATE tRNA. ii 217

DIXON (personal communication). It modifies specifically U and G and prevents pancreatic ribonuclease from hydrolysing the sequences Up -> Xp. The chemical modification and the pancreatic ribonuclease digestion are described under MATERIAL AND METHODS. The modified oligonucleotides were separated as indicated in Fig. 5. The longest oligonucleotides were analysed. Among them Peak X was the most interesting one. It was desalted, treated during 2 h with i ml of 12 % NH4OH to remove the blocking groups, evaporated to dryness and submitted to a high voltage electrophoresis (Fig. 6). The oligonucleotide called X A (Fig. 6) was eluted as previously described 5 and hydrolysed with 15/zg of pancreatic ribonuclease in 50/zl of o.I M triethylammonium bicarbonate. The hydrolysate was submitted to a high voltage electrophoresis performed as in Fig. 6. I t gave two spots XA 1 and XA 2. They were eluted and analysed. The following sequences were found: XA 1 was the tetranucleotide G-G-G-Cp and XA 2 the pentanucleotide A-G-A-A-Up. This result shows that the sequence A-G-A-A-U-G is followed by G-G-C at the 3' end and yields the following sequence to Fragment AI: C-A-G-A-A-U-G-G-G-C-G-Cp.

- - X A --I~,,- ! i i ; . d r

B l u e I m

origin

Fig. 6. High voltage e|ectrophoresis on DEAE-cel]u]ose paper of o|igonucleotide X derived f rom DEAE-ce l lu lose c o l u m n (Fig. 5). The e lec t rophores is was pe r fo rmed in 7 % formic acid wi th a vo l tage of 7o0 V for 18 h.

Fragment A 2 of ]31g. 3 gave, after T 1 ribonuclease hydrolysis, the following oligonucleotides in equimolar ratio: U-Gp, A-U-A-Gp, pU-C-C-Gp and U-U-~-A-A- hUp. The pU-C-C-Gp sequence must be at the 5' end of this fragment coming from the 5' terminal end of the tRNA Asp. It is followed by the U-Gp fragment as will be shown below (Fragment 3 of Fig. 7), U-U-k~r-A-A-hU must be at the 3' end since Fragment A 2 derives from a pancreatic ribonuclease digestion of tRNA Asp. Therefore A-U-A-Gp must be between pU-C-C-G-U-Gp and U-U-~O'-A-A-hUp and the primary structure of Fragment A 2 is pU-C-C-G-U-G-A-U-A-G-U-U-~-A-A-hUp.

Fragment Blof Fig. 4 gave the following products in equimolar ratio: Cp, Gp, C-Gp, A-Gp, U-C-Gp and A-A-U-U-C-C-C-C-Gp. The presence of A-A-U-U-C-C-C-C- Gp places this fragment in the half of tRNA ASp bearing the 3' terminal end (Frag- ment II in Table I). A-A-U-U-C-C-C-C-Gp must be at the 5' terminal end of Frag- ment B1 because, in the nonadecanucleotide found among the exhaustive digestion products of tRNA Asp with T 1 ribonuclease 1, it is preceeded by T-~-C. The 3' terminal end of Fragment B~ is Cp because B~ comes from a partial pancreatic ribonuclease digestion of tRNA Asp. Analysis of Fragment 6 (see below) will show that the sequence U-C-Gp is on the 3' end of the fragment A-A-U-U-C-C-C-C-Gp. Therefore Gp, A-Gp and C-Gp must be placed between U-C-Gp and Cp. They can only be a part of the 3' terminal end of the 3' half of the molecule of tRNAAsp: G-G-A-G-Cp. The structure of Fragment 131 is therefore A-A-U-U-C-C-C-C-G-U-C-G-C-G-G-A-G-Cp.

Fragment B 2 of Fig. 4 gives, after a complete T 1 ribonuclease digestion, the following mono- and oligonucleotides Gp, T-~Pp, U-Gp, C-C-A-Gp and A-U-mSC-Gp


218 j. GANGLOFF et al.

in the molar ratios 4 : I : I: I : I. This fragment is a part of the oligonucleotide of 23 nucleotides above mentioned and called Fragment a: C-C-A-G-A-U-msC-G-G-G - G-T-~-C-A-A-U-U-C-C-C-C-Gp. The dinucleotide U-Gp and the Gp can only be placed at the 5' terminal end of Fragment B 2. The pancreatic ribonuclease digestion of Fragment B 2 gives a dinucleotide G-Up; therefore the following structure can be given to Fragment B2: G-U-G-C-C-A-G-A-U-mSC-G-G-G-G-T-hep.

Fragment B s of Fig. 4 gives the following mono- and oligonucleotides in equimolar ratio after a complete T 1 ribonuclease digestion: hUp, U-Gp, A-U-A-Gp, pU-C-C-Gp, U-U-~V-A-A-hU-Gp. This is Fragment A 2, bearing at the 3' terminal end a sequence G-G-hUp.

Results o~ partial hydrolysis with T 1 ribonuclease. The elution pat tern obtained upon chromatography of a partial T 1 ribonuclease digest of tRNA Asp on DEAE- cellulose is shown in Fig. 7. Peaks 3, 5 and 6 contained large fragments essential for sequence determination of the tRNA.

1.5

I I 150

,10

0 50 100

E = o QS"

J

I 200 250

F r Q c t i o n s o f 4 .8 m l

G t~ Z

Fig. 7- DEAE-cellulose column (0. 7 cm × 200 cm) ch roma tography of a part ial ribonuclease T 1 digest of t R N A Asp (20 mg) Elut ion with a linear gradient (18oo ml total volume) of NaCI (o to 0.5 M) in 0.02 M Tris (pH 7-3)-7 M urea.

Peak 3 of Fig. 7 was homogenous on rechromatography on DEAE cellulose at pH 3. I t gave, after exhaustive T 1 ribonuclease digestion, two fragments: pU-C-C-Gp and U-Gp which were separated by high voltage electrophoresis. These results clearly indicate that Peak 3 contains the following hexanucleotide: pU-C-C-G-U-Gp derived from the 5' terminal end of the tRNA Asp.

Peak 5 of Fig. 7 was freed from its minor contaminants by chromatography on DEAE cellulose at pH 3- I t gave after desalting ar.d exhaustive T1 ribonuclease digestion the following three oligonucleotides U-Gp, A-U-A-Gp and pU-C-C-Gp, indicating that the tRNA Asp has the sequence pU-C-C-G-U-G-A-U-A-Gp at the 5' terminal end.

Peak 6 of Fig. 7 was rechromatographed on DEAE-cellulose at pH 3 and so freed of minor contaminants. I ts T 1 ribonuclease digest, submitted to a high voltage

Biochim. Biophys. Acta, 259 (1972) 2IO-222

P R I M A R Y S T R U C T U R E O F Y E A S T A S P A R T A T E tRNA. II 219

electrophoresis, gave the following nucleotides and oligonucleotides: Gp, U-C-Gp, A-U-mSC-Gp and T-~r/-C-A-A-U-U-C-C-C-C-Gp in the molar ratio of 3 : I : I : I. The mononucleotides Gp, the tetranucleotide A-U-mSC-Gp and the dodecanucleotide are included ii1 the oligonucleotide a: C-C-A-G-A-U-mSC-G-G-G-G-T-~/AC-A-A-U_U_C_C - C-C-Gp. Therefore the trinucleotide U-C-Gp can only be placed at the 3' terminal end of the oligonucleotide of Peak 6, the sequence of which is: A-U-mSC-G-G-G-G - T-~-C-A-A-U-U-C-C-C-C-G-U-C-Gp.

Derivation o] the complete sequence Sequence o] the 5' terminal hall o] tRNA Asp. The tetranucleotide hU-C-A-Gp

obtained by total T 1 ribonuclease digestion of the 5' terminal half of tRNA Asp overlaps with Fragments B 3 and A~ (Table I I ) and permits the construction of a fragment of 31 nucleotides. This fragment accounts for almost all the nucleotides of the 5' half of the molecule of tRNA Asp except for ~ -U-Gp which therefore must terminate, on the 3' end, the 5' terminal half of the molecule of tRNA Asp (Table II) .

T A B L E II

D E R I V A T I O N OF THE COMPLETE S E Q U E N C E OF F R A G M E N T I OF t R N A A~p FROM LARGE OLIGONU- CLEOTIDES O B T A I N E D FROM PARTIAL DIGESTIONS W I T t I T1 A N D PANCREATIC R I B O N U C L E A S E

Digestion Fragment Sequence

Part ia l pancrea t ic r ibonuclease

Par t ia l T 1 r ibonuclease

Complete sequence of I

A1 (Fig- 3) A 2 (Fig. 3) 13 3 (Fig. 4) 3 (Fig. 7) 6 (Fig. 7)

CAGAAUGGGCGCp pUCCGUGAUAGUU~r/AAhUp pUCCG UGAUAGUUl t /AAhUGGhUp pUCCGUGp p U C C G U G A U A G p

p U C C G U G A U A G U U ~ A A h U G G h U C A G A A U G G G C G C ~ U G p

Sequence o[ the 3' terminal hall o] t R N A asp. Table I I I shows tha t Fragments B 1 and B 2 can be overlapped by the sequence of 23 nucleotides of Fragment a, and this permits construction of a sequence of 35 nucleotides. This sequence accounts for almost all the nucleotides of the 3' terminal half of tRNA Asp that we called

T A B L E I I I

D E R I V A T I O N OF T H E COMPLETE S E Q U E N C E OF F R A G M E N T II OF t R ~ q A Asp FROM LARGE O L I G O N U - CLEOTIDES O B T A I N E D FROM PARTIAL DIGESTIONS W I T H T 1 A N D PANCREATIC R I B O N U C L E A S E

Digestion Fragment Sequence

Part ia l pancrea t ic B 1 (Fig. 4) r ibonuclease ]3~ (Fig. 4)

Par t ia l T 1 a* r ibonuclease 6 (Fig. 7)

Complete sequence of I I

UCmXG CGUGCCAGAUmSCGGGGT~/CAAUUCCCCGUCGCGC, AGCCA

AAUUCCCCGUCGCGGAGC GUGCCAGAUmSCGGGGTkU CCAGAUmsCGGG GT~rXCAA UUCCCCC- AUmSCGGGGTt//CAA UUCCCCGU CG

* The s t ruc ture of this f r agment was de te rmined as indicated in RESULTS.


220 J. GANGLOFF el al.

Fragment I [ in Table I except for U-C-mlGp and Cp belonging to the sequence U-C-m~G-Cp. Therefore, the 3' terminal half of tRNA A~p is terminated on the 5' end by the sequence U-C-mlG-Cp.

Complete sequence. The 3' and the 5' halves of the molecule can be assembled and the 3' terminal half completed by the sequence CCA to give the total sequence, which can be arranged in the cloverleaf model shown in Fig. 8. In addition to the structural properties described in the preceeding paper 1 (75 nucleotides including 8 minor ones, pUp as the 5' terminal end, G-C-C-A as the 3' terminal end, G-T-~V-Cp sequence), other structural features can be mentioned:

AoN C C G

pU-A C-G C-G G-C U-G G-C A-U U A

• u G C C C C u A ~ ' ' ' ' ' A

m C G G G G ~uAA~uuG UA T~C G h U c A G A A U G A G

G-C G-C C-G G-U C-G

~J C U rnlG G u C

Fig. 8. Cloverleaf model of tRNA A'p.

All tRNAs of known structure have a U or a derivative of U in the eighth position from the 5' end. This is also the case with tRNA Asp.

Aspartate tRNA has an AAhU sequence instead of the AGhU sequence com- mon to most sequenced yeast tRNAs. CRAMER et al. 6 have proposed that this AG sequence could give hydrogen bonds with the sequence ~vC of GT~C. This would not be possible in the case of tRNA Asp. But this tRNA could take the tertiary structure proposed by LEVITT 7 with a base pair between A15 and U4~. Since the determination of the structure of tRNA Asp (ref. 8 and 9), five other structures of tRNA showing a sequence AAhU have been determined: tRNA Leu of Escherichia coli l°,n, tRNA Trp of E. coli 12, tRNA Trp of brewer's yeast 13 and tRNAA/I g of brewer's yeast 14. These five tRNAs present also a U at the place corresponding to the U,~ of tRNA Aw.

The sequence G-G found in all tRNAs of known sequence in Position 17 and 18 is also present in tRNA A~p.

m~C is the 28th nucleotide from the 3' end as in tRNA Phe (ref 15) and in tRNA val (ref. 16) of yeast, whereas it is the 29th in the other tRNAs containing this nucleotide. In tRNA phe, tRNA val and tRNA A~p, this mSC is paired with a G: this is one of the 5 base pairs in the region sustaining the T~C loop.

There are seven base pairs in the stem bearing the 5' and 3' ends of the molecule as in all the sequenced tRNAs except tRNA ~Met of E. coli 17. There are five base


PRIMARY STRUCTURE OF YEAST ASPARTATE tRNA. II 221

pairs in the stems sustaining the T ~ C and the anticodon loops, which is a general feature. The presence of five G • C pairs in the stem sustaining the T ~ C loop gives to this par t of the molecule a great stability towards T 1 ribonuclease digestion.

However, the stem sustaining the hU loop has four base pairs but no G • C pair. In all tRNAs of known sequence, the first and the last base pairs in this stem are G . C pairs; tRNA Asp is the only exception, having a ~ . G and a G • U pair, respectively in these positions. I t might be that this feature confers a low stability to this part of tile molecule.

In the different stems there are four exceptions to the classical G • C and A • U base pairs, namely 3 G • U pairs and I G • ~ pair.

The different loops show the following lengths: 7 nucleotides for the anticodon and the T ~ C loops, as in all sequenced tRNAs, 8 nucleotides for the hU loop, whose length varies between 8 and I I nucleotides. The extra arm is exceptionally short: it is only 4 nucleotides long.

Among the potential anticodons are two GUC, one GhUC and one AUmSC sequences. One GUC sequence and the m~C of the AUmSC oligonucleotide are placed in base-paired regions. Of the two remaining possible triplets the location of GUC in tRNA Asp is very similar to the locations of presumed anticodons in other tRNAs. This GUC is also present in a loop and its nucleotides are, respectively, the third, fourth and fifth base in the loop. As in all known cases this presumed anticodon is preceded by uridine. This is also the case of tRNA Asp. The base following the anticodon is a modified G. Finally, all tRNAs containing G in the anticodon sequence are highly susceptible to T 1 ribonuclease a t tack in this region; this is also the case for the presumed anticodon GUC in aspartate tRNA. This sequence could correspond to the aspartate codons, GAC and GAU, according to the wobble hypothesis is.

ACKN OWLEDGEMENTS

The authors thank Mrs. M. L. Gangloff and N. Menard for their skilful assis- tance and Mrs Schlegel for the fractionation of tRNA by countercurrent distribution. This work was supported in part by grants from C.N.R.S. (L. A. No. 119) from the Delegation Generale ~ la Recherche Scientifique et Technique (Convention de Biologie Mol~culaire) and from the Commissariat ~t l 'Energie Atomique.

R E F E R E N C E S

I J. GANGLOFF, G. KEITH, J. P. EBEL AND G. DIRHEIMER, Biochim. Biophys. Acta, 259 (1972) 198.

2 S. H. CHANG AND U. L. RAJ BFIANDARY, J. Biol. Chem., 243 (1968) 592. 3 D. DUTTING, H. FELDMANN AND H. G-. ZACHAU, Hoppe Seyler's Z. Physiol. Chem., 347 (1966)

249. 4 p. T. GIL~.M, J. Am. Chem. Soc., 84 (1962) 687. 5 J. GANGLOFF, G-. KEITH AND G. DIRHEIMER, Bull. Soc. Chim. Biol., 52 (197 o) 125. 6 F. CRAMER, R_. DOEPNER, F. VAN DE HAAR, E. SCHLIMME AND H. SEIDEL, Proc. Natl. ,4cad.

Sci. U.S., 61 (1968) 1384. 7 M. LEVITT, Nature, 224 (1969) 759. 8 G-. I~EITH, J. G-ANGLOFF, J. P. EBEL AND Cr. DIRHEIMER, C. R. Acad. Sci. Paris, 271 (i97o) 613. 9 J- GANGLOFF, G'. KEITH, J. P. EBEL AND Cr. 1)IRHEIMER, Nature New Biol., 23o (1971) 125.

1o S. K. DUBE, K. A. 1V[ARCKER AND A. YUDELEVICH, F E B S Lett., 9 (197 o) 168. 71 H. U. BLANK AND D. SOLL, Biochem. Biophys. Res. Commun., 43 (1971) 1192.


222 j . GANGLOFF et al.

12 D. HIRSCH, Nature, 228 (197 o) 57. 13 O. I~EITH, A. ROY, J. P. EBEL AND G. DIRHEIMER, F E B S Lett., 1971, 17 (1971) 306. 14 B. KUNTZEL, J. ~¥EISSENBACH AND G'. DIRHEIMER, F E B S Lett., 1972, s u b m i t t e d for publ i -

cat ion. 15 U. L. I{AJ BHANDARY, S. H. CHANG, H. J. GROSS, F. HARADA, F. KIMURA AND S. •ISHIMURA

Fed. Proc., 28 (1969) 409 • 16 J. BONNET, J. P. EBEL AND G. DIRHEIMER, F E B S Lett., 1971, 15 (1971) 286. 17 S. CORY, S. K. DUBE, ]3. F. C. CLARK &ND K. A. ]~ARCKER, Nature, 220 (1968) lO39. 18 V. H. C. CRICK, J. Mol. Biol., 19 (1966) 548.

Biochim. Biophys. Acta, 259 (1972) 21o 222

the primary structure of aspartate transfer ribonucleic acid from brewers yeast: ii. partial...

Documents