Eur. J. Biochem. 24 (1971) 296-302

Substrate Properties of Yeast tRNAPhe Oxidized and Reduced at the 3'-Terminal Ribose

Friedrich VON DER HAAR, Eckhard SCHLIMME, Manuel G~MEZ-GUILLEN, and Friedrich CRAMER Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen

(Received August 16/0ctober 4, 1971)

Native yeast tRNAPhe and this tRNAPhe with the 3'-terminal AMP removed were oxidized by NaIO, and subsequently reduced by NaBH,. The following investigations were undertaken with these substrates: aminoacylation in borate buffer and measurement of the life-time of the aminoacylated oxidized an reduced tRNA, hydrolysis with snake-venom phosphodiesterase and pyrophosphorolysis with tRNA nucleotidyl transferase.

Whereas the oxidized and reduced yeast tRNAPhe is a good substrate for the enzymes investigated, the corresponding tRNAPhe with the 3'-terminal AMP removed is a very poor, if any, substrate in these reactions. From the data obtained and from proton-magnetic-resonance investigations with oxidized and reduced AMP and ATP it is concluded that the removal of the C2'-C3' bond and introduction of two hydrogen atoms instead distorts the original ribose con- formation a t the 3'-terminus. Under the assumption that this distortion is different for C,, and A,, the difference in reactivity between the two oxidized and reduced tRNAs can be ex- plained.

Yeast tRNAPhe with a ring-opened ribose a t the 3'-end (tRNAPhe-C-C-Ao,i.red), generated by perio- date oxidation and subsequent borohydride reduction, can still be aminoacylated with the same specificity and to the same extent as unmodified yeast tRNAPhe [I]. Recently Chen and Ofengand found that a tRNA thus modified is not a substrate in protein synthesis [2].

This paper describes the aminoacylation in borate buffer compared to Tris buffer, the degradation with snake-venom phosphodiesterase and the pyrophos- phorolysis with tRNA nucleotidyl transferase of yeast tRNAP"-C-C-Aoxi.red and tRNAPhe-C-Coxi.i-ed.

MATERIALS AND METHODS

Materials Bakers' yeast tRNAPhe was purified from

tRNAb"1k (Boehringer Mannheim GmbH, Mann- heim, Germany) [3] up to a degree of aminoacylation of 1550 pmol phenylalanine per AZ6,, unit tRNA.

Unusual Abbreviations. tRNAPhe-C?,-C75-A76, native tRNAPhe (phenylalanine transfer ribonucleic acid) ; tRNAPhe C-C, tRNAPhe lacking the 3'-terminal AMP; tRNAPhe- C-C-A4-oxi-red, tXNAPhe with 2'C-3'C linkage of the 3'- terminal ribose split by periodate-oxidation and subsequent- ly reduced with borohydride; Phe-tRNAPhe, phenylalanyl- tRNAPhe.

Enzymes. Phenylalanyl-tRNA synthetase (EC 6.1.6.-) ; snake-venom phosphodiesterase (EC 3.1.4.1) ; tRNA nucleo- tidy1 transferase (EC 2.7.7.).

Definition. A,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 a t 260 nm, when measured in a 1-cm pathlength cell.

Phenylalanyl-tRNA synthetase was purified from bakers' yeast up to a specific activity of 316 units/mg [4] ; I unit = incorporation of 1 nmol phenylalanine into tRNAPhe per minute.

Snake-venom phosphodiesterase (1 mg/ml) was a product of Boehringer Mannheim GmbH (Mann- heim, Germany).

tRNA nucleotidyl transferase was purified from bakers' yeast to a specific activity of about 400 units/mg. This is the product obtained by CM- cellulose chromatography during purification [5] ; 1 unit = incorporation of 1 nmol ATP into tRNAPhe- C-C per minute.

14C-labelled amino acids (50 mCi/mmol), [14C]- ATP (15.8 pmol/ml, 32 pCi/pmol), [3H]ATP (5 mCi/ 10 ml, solution containing 50°/, ethanol, 14.4 Ci/ mmol) and [14C]CTP (9.2 pmol/ml, 21.8 pCilpmo1) were purchased from Schwarz Bioresearch (Orange- burg, U.S.A.). The [3H]ATP solution was evaporated to dryness and redissolved in twice-distilled water. Unlabelled ATP was added to give a concentration of 6 pmol/ml containing 2.5 mCi/ml.

METHODS

Oxidation and Reduction Oxidation and reduction of yeast tRNAPhe-

C-C-A and tRNAPhe-C-C to tRNAPhe-C-C-Aoxi-red and tRNAPhe-C-Coxi.red were carried out as described

Vo1.24, No.2,1971 F. VON DEF~ HAAR, E. SCE~LIMME, M. G~MEZ-GUILLEN, and F. ~ A M E R 297

Aminoacylation Aminoacylation of native and modified yeast

tRNAPhe were tested in the aminoacylation assay previously described [4].

In the case of aminoacylation in borate buffer, 0.15 M borate buffer pH 7.6 was employed instead of Tris-buffer; all the other components remaining the same.

For preparation of Phe-tRNAPhe-C-C-A and Phe-tRNAPhe-C-C-AoXi-red 5 A,,, units of tRNA were aminoacylated in 0.5 ml of the reaction mixture de- scribed [4]. The reaction was stopped by addition of 0.15 ml of 2 M sodium-acetate buffer pH 4.5. Phe- tRNA was separated from other materials by passage over a 1 x 20 cm Sephadex-G-25 column, using 0.01 M acetic acid as eluant. The material appearing in the void volume was collected and freeze dried.

Half-life of these aminoacyl tRNAs was determin- ed by measuring the release of [14C]phenylalanine in 0.1 M Tris-HC1 buffer pH 7.6 a t 22 "C as in the amino- acylation assay [4].

Xnake- Venom Phosphodiesterase Degradation Degradation of native and modified yeast tRNAPhe

with snake-venom phosphodiesterase was performed according to the method of Zubay [6] a t 20 "C in 0.01 MTris-HClbuffer pH 7.6 containing 0.01 M Mg2+. 20mg yeast tRNAPhe were incubated with 200pg phosphodiesterase in 12 ml of the reaction mixture for 6 h. The solution was then passed over a DEAE- cellulose column (3 x 5 cm) which was washed suc- cessively with twice-distilled water and 0.4 M NaC1. The tRNA was eluted with 1 M NaC1, dialysed, and lyophilized.

Dependence of the Xnake- Venom Phosphodiesterase Degradation

on Time and Amount of Enzyme Samples of tRNAPhe-C-C-[3H]A, tRNAPhe-C-C-

['HIAoxi-red, tRNAPhe-[14C]C-[14C]C and tRNAPhe- [14C]C-[14C]Coxi-red (prepared as described below) were digested in 100 pl of the reaction mixture. Aliquots were withdrawn a t certain intervals and ap- plied to Whatman-3 MM filter strips. The strips were chromatographed in the system isopropanol- 1 M- sodium acetate pH 5-ammonium sulfate, saturated at 20 "C (1 : 10 : 1, v/v/v). The radioactive label a t the origin was measured in a liquid-scintillation counter (Tricarb 3375). If unlabelled products were tested, aliquots were worked up as in the preparative snake- venom phosphodiesterase degradation and tested by incorporation of either labelled CTP or ATP by tRNA nucleotidyl transferase. Specifications of en- zyme and tRNA concentrations as well as reaction time are given under Results. 20 Eur. J. Biochem., Vol. 2-1

Regeneration with tRNA- Nucleotidyl Transf erme

Incorporation of labelled and unlabelled ATP and CTP into the 3'-end of tRNA was performed as described [l]. The incubation mixture contained 0.15 M Tris-HC1 buffer pH 9, 0.05 M K+, 0.01 M Mg2+ and 1 pg tRNA nucleotidyl transferase per 5-10A2,, units tRNA. The molar ratio of tRNA to nucleoside triphosphate, NTP, in the reaction mix- ture was about 1 : 25. The regeneration was carried out a t 32 "C for 60 min.

tRNAPhe-C-C-[3H]A was prepared from phospho- diesterase-degraded tRNAPhe by incorporation of CTP and [3H]ATP. tRNAPhe-[14C]C-[14C]C was pre- pared by incorporation of [14C]CTP into phospho- diesterase-degraded tRNAPhe omitting ATP. The regenerated tRNA was isolated as described for the preparation of phosphodiesterase-degraded tRNAPhe. The labelled products tRNAPhe-C-C-[SH]A and tRNAPhe-[W]C-[l4C]C as well as tRNPhe-C-C-[3H] Aoxi-red and tRNAPhe-[14C]C-[14C]Coxi-red were cha- racterized by alkaline hydrolysis (0.3 M KOH a t 36 "C for 18 h) followed by paper chromatography (Paper 2043 b, Schleicher & Schull, Dassel, Germany) in the system ethanol-1 M ammonium acetate (8:2, v/v). The paper chromatograms were cut into pieces of 1 cm length and the radioactive label was measured in a tricarb scintillation-counter. The Rg values of the labelled products are: [3H]A 0.48 [l],

and [14C]CMP 0.12. ['H]Aoxi.red 0.58 [I], [l4C]c 0.58, [14C]Coxi-red 0.69,

Pyro phosphorolysis with tRNA-Nucleotidyl Transferase

Pyrophosphorolysis with native and modified tRNAPhe was carried out a t 32 "C in 0.1 M cacodylate buffer pH 7.6, containing 0.01 M Mg2+, 0.03 M K+ and 0.7 mM pyrophosphate in a total volume of 100 p1. The dependence of reaction time and amount of enzyme was analyzed in the same way as described above. Concentration of tRNAPhe and tRNA nucleo- tidy1 transferase as well as the reaction time are spe- cified in Results.

Preparation and Identification of ATPozt-red

1 - (9-adeny1)- 1 '- (triphosphoryl-oxy- methyl)-diethyleneglycol, was prepared by periodate oxidation followed by borohydride reduction ac- cording to the procedure of Khym [7]. 1.5 mmol ATP was dissolved in 20 ml of 0.1 M NaIO, and 10 ml 0.1 M phosphate buffer pH 7.0 was added. To destroy excess NaIO, 4 ml of 1 M ethyleneglycol was added 20 min later. The solution was diluted to 80 ml with twice-distilled water and 500 mg of NaBH, was ad- ded. After 16 h excess NaBH, was destroyed by ad- ding 25 ml 1 M formic acid. The solution was neutra-

ATPoxi-red,

298 Substrate Properties of Modified Yeast tRNAPhe Eur. J. Biochem.

lized with 25 ml 1 M ammonia. The total reaction mixture was diluted up to 400ml. Adenosine and AMP were oxidized and reduced in the same way.

An aliquot of the reaction mixture containing 6500 A,,, units was applied to a DEAE-cellulose column (Whatman DE 11, HC0,- form, 4 0 ~ 1.5 cm). After washing with 1 1 of twice-distilled water, ATPoxi-red was eluted with a h e a r gradient of tri- ethylammonium bicarbonate (mixing chamber 3 1 twice-distilled water, second chamber 3 1 0.5 M NHEt,HCO,). The ATPoxi.red-contahhg fraction was determined by electrophoresis and lyophilized. The amorphous NHEt,-salt (3730 AZ6, units, approx. 5701, yield) was transformed to the Na+ salt by pas- sage over an ion-exchange column (Merck I , Naf form, Merck Darmstadt, Germany). The final product was characterized by ultraviolet spectroscopy, (Am,, : 259 nm, E = 14800) and electrophoresis on paper 2043b (Schleicher & Schiill, Dassel, Germany) in 0.05 M ammonium formate buffer pH 3.5 for 1.5 h at 1500 V. Mobilities: AMP = 1, ADP = 1.9, ATP = 2.5, ATPoxi-red = 2.3, ADPoxi-red = 1.8. The final product was a mixture of ATPoxi-red and ADPoxi-red ; the ratio being ATPoxi-red/ADPoxi-red = 4: 1, as determined by ultraviolet spectroscopy after elution of the spots.

Elementary analysis calculated for a mixture of

Calculated: C 18.55; H 3.42; N 10.82; P 13.35. Found: C 18.73; H 3.47; N 10.77; P 13.54O/,.

Proton-magnetic-resonance spectra were taken on a Varian HA-100 in 2H,0 with trimethylsilyl- propane sulfonic acid as internal standard ; the data are given in Results.

ADPoxi-red and SOo/, ATPOxi-red) 4 HZO;

RESULTS Oxidation and Reduction of tRNAPhe

Oxidation with periodate and subsequent reduc- tion with borohydride leads to a tRNA with a ring- opened terminal ribose [l] :

tRNAPhe-C -C -CH y p HQ+ iq$+

P h e - tRNA syn thet ase I

-- P h e O Q I Phe - tRNA

synthe tase

Aminoacylation of tRNAPhe- C- C-Aozf-red in Borate Buffer and Stability of Phe-tRNAPhe- C- C-Aozi-red

tRNAPhe-C-C-A is enzymatically aminoacylated in borate buffer s igdcant ly slower than in Tris-

.- XI

400 P- A' u

0 10 20 30 0 10 20 30 ~ ~. Time (min)

Fig.1. Aminoacylation of 0.12 A,,, unit (A) tRNAPhe-C-C-A and ( B ) tRNAPhe-C-C-A.,t-red in Tris buffer ( A ) and borate buffer (x ) using 1 pg of phenylalanyl-tRNA-synthetase prep-

aration

Table 1. K,-values and mximum velocity of native and oxidized-reduced tRNAPhe in borate and Tris buffer

The values were calculated from the data obtained by the aminoacylation assay [4] with the help of a computer pro-

gram written according to [9]

tRNA Buffer R, Relatives V

tRNA"e-C-C-A Tris 0.88 100 borate 0.99 15

tRNAPhe-C-C-A,,i.,,d Tris 1.2 25 borate 0.92 15

a Aminoacylation of tRNAPhe-C-C-A in Tris buffer was taken as 100 "Io.

buffer, all other conditions being identical (Fig. 1, Table 1). No such decrease is observed in the case of tRNAPhe-C-C-Aoxi.red (Fig. 1, Table 1) suggesting that the 2' and 3' hydroxy group no longer form a borate complex of the same stability, presumably because of distortion of the hydroxy groups. This interpretation is supported by the fact that during electrophoresis in borate buffer the following relative mobilities are found : AMP = 1 .O ; AMPoxi-red = 0.78 ; Ado = 0.37 ; AdOoxi-red = 0. This shows that also the monomer AdOoxi-red does not form a borate complex

Similar information can be derived from the mea- surement of the life-time of Phe-tRNAPhe-C-C-A and Phe-tRNAPhe-C-C-A,,i.red. In Tris buffer pH 7.6 a t room temperature the half-life of Phe-tRNAPhe- C-C-A is 41 min whereas under the same conditions the half-life of Phe-tRNAPhe-C-C-Aoxi-red is 102 min.

P I .

Degradation of tRNAPhe with Snake- Venom Phosphodiesterase

Phosphodiesterase degradation of tRNAPhe-[14C]C,4- [14C]C7,-[3H]A76. tRNAPhe-[14C]C-[14C]C-[3H]A was treated with varying amounts of phosphodiesterase using otherwise identical conditions according to [6]. The half-life of the 3'-terminal A,, is about 4min

Vo1.24. No.2,1971 F. VON DER HAAR, E. SCELIMME, M. G~MEZ-GUIUEN, and F. CRAMER 299

\ '!I ' 20 ' 4b ' 60 ' 80 '100120 160 200 240 280 320 360

Time (rnin)

160 200 240 280 320 360 Time (min)

Fig.2. Releme of ( A ) ISHIAMP and ( B ) [14C]CMP from tRNAphe-[14C]C-[14C]C[SH]A using differing snake-venom- phosphodiesterase concentrationa. Total reaction mixture loop1 containing 0.3 A,,, unit tRNA. The ratio of tRNA

using 1.5 pg enzyme per Ate, unit tRNA. Under the same conditions about 50°/, of the total label of C,, and C,, is removed within 80 min. Then the reaction ceases. Assuming that a t this point only C,, is removed the half-life of C,, is about 20 min.

The latter finding differs from the preparative treatment of tRNAPhe in mg quantities. Phospho- diesterase removed the total 3'-terminal C-C-A end from tRNAPhe, using the conditions given by Zubay [6] for preparation of bulk tRNA-C. This result was checked in the following way. Phosphodiesterase- treated tRNAPhe was regenerated with [14C]CTP using tRNA nucleotidyl transferase. This tRNA was hydrolyzed with alkali, and the nucleosides end nucleotides were separated by paper chromatog- raphy. The ratio of [W]CMP to [14C]C from tRNAPhe- [14C]C-[14C]C (48500 counts x min-l x A,,,-unit-l) was 1688 to 1521 counts/min. 20'

to enzyme was as follows: x , 1 A,,, unit tRNAPhe: 0.25 pg enzyme; 0, 1 A,,, unit tRNAPhe: 0.5 pg enzyme; 0, 1 A,, unit tRNAPhe: 1 pg enzyme; A , 1 A,,, unit tRNAPhe:

3 pg enzyme; 0, 1 A,,, unit tRNAPhe: 7.5 pg enzyme

This discrepancy between the preparative and analytical phosphodiesterase treatment shows that the degradation of tRNA by phosphodiesterase has to be controlled very carefully. Fig.2 gives the results for the removal of c,,, c,, and A,,, as increasing amounts of phosphodiesterase are used.

Comparison of Phosphodiesterase Degradation oi Unmodified and Modified tRNAPhe. Table 2 gives the results of phosphodiesterase treatment of tRNAPhe- C-C-[3H]A and tRNAPhe-[14C]C-[14C]C compared to tRNAPhe-C-C-[3H]A,xi-~e~ and tRNAPhe-[14C]C- [l'C]Coxi-red. Since the reaction velocities are rather different for the four derivatives, it was not possible to find reaction conditions under which the reactivity of the four substrates can be compared directly. Using an amount of 33 pg enzyme/A,,, unit tRNA the A?, as well as C,, and C,, are removed up to 9501, within 5 min. Under these conditions the A,,

300 Substrate Properties of Modified Yeast tRNAPhe Eur. J. Biochem.

Table 2. Release of 3’-temninul nucleotidea of yea& tRNAPhe by differing amounts of snake-venom p?wsp?wdiesterase

Substrate (0.05 Asso mitl1OO 14

~~ - Intact 3‘-terminus after

5 min 120 min Enzyme Monitored base

“lo O/O

tRNAPhe-C-C-[’H]A 0.16 87 28 0.64 27 2

33 5 3

tRNA”e-C-C-[’H]Ao,i.~e~ 1.6 85 79 33 74 40

100 65 47

tRNAPhe-[14C]C-[14C]C 0.5 c74 + c75 70 19 33 c74 + (775 5 4

tRNA”e-[14C]C-[‘4ClCo~-~e~ 0.5 (374 + c75 99 99 33 c74 + c75 94 93

lrg/Ass0 unit tRNA

c75 97a 96.58 83.5 c74 + (375 85 87

c75 938 948 ~

* The velocity-determining step is the hydrolysis of CTS oxl-red. In order to calculate the value of removal of C75 oli-red, half the amount of radio- activity released has to be taken.

is removed to the extent of 26O/, within 5 min and to 60°/, within 120 min. The c,, oxi-red, however, is more than 95O/, stable after 120min under these conditions and even if the amount of enzyme is raised to 83.5 pg/AZso unit tRNA there is no significant in- crease in the release of c,, oxi-red.

tRNA -Nucleotidyl- Transf erase Action

tRNA nucleotidyl transferase catalyses the re- action :

tRNA . . . . + 2 CTP + 1 ATP + tRNA-C-C-A + 3 PPi

Whereas the nucleotidyl transfer is well known [lo- 151 , the pyrophosphorolysis reaction is less thoroughly investigated. In the pyrophosphorolysis reaction at higher pyrophosphate concentrations Mg,+ salts of pyrophosphate are precipitated. 0.1 M caco- dylate buffer pH 7.6 containing 0.01 M Mg2+ is sui- table up to a pyrophosphate concentration of 0.7 mM. Pyrophosphorolysis seems to proceed more slowly than the incorporation of nucleoside triphosphate into tRNA. Therefore a higher amount of enzyme (20 to 50-fold) has to be used to follow the reaction in a measurable time range.

In the pyrophosphorolysis reaction by tRNA nucleotidyl transferase, tRNAPhe-C-C-Aoxi-red is a less reactive substrate than unmodified tRNA (Fig. 3). Since tRNA nucleotidyl transferase is slowly inactivated during incubation, after a reaction time of about 1 h additional enzyme was added to the mix- ture. Pyrophosphorolysis started again immediately after adding active tRNA nucleotidyl transferase. Pyrophosphorolysis of tRNAPhe-[14C]C-[14C]C~xi-red is achieved only to a very poor extent compared to tRNAPhe-C-C-AoM-red and tRNAPhe-C-C. The data

of pyrophosphorolysis of the several modified pro- ducts compared to starting material are summarized in Table 3.

ATPoxi-red could not be incorporated into tRNAPhe-C-C instead of ATP. Since no labelled ATPoxi-red was available, this reaction was tested by incubation of tRNAPhe-C-C with ATPoxi-red in the regeneration mixture, isolation of incubated tRNAPhe by column chromatography and a subsequent test of aminoacylation of this modified tRNAPhe. This product was not capable of being aminoacylated. Therefore, an incorporation of ATPoxi-red did not take place, since tRNAPhe-C-C-Aoxi-red is fully amino- acylated [l]. Incorporation of [3H]ATP into tRNAPhe- C-Coxi-red could not be achieved either.

Conformation of ATPozt.red Table 4 gives the data of proton-magnetic-reso-

nance spectra of AMPofi-red and ATPoxi-red. The pro- tons of H3’, H4’ and H5’ can not be distinguished be- cause they appear as a multiplet around 6.15ppm. The main information comes from the protons of C1’ and C2’. The signals of the protons of C2’ are of an A, type, and that of Cl’ is of an AX, type, showing a coupling constant of 5 Hz in AMPoxi-red and 4.5 Hz in ATPoxi-red. This indicates that a free rotation around the Cl‘-C2’ linkage is possible. Otherwise one would expect an ABX spectrum rather than an A, spectrum for the C2’ protons, and furthermore the coupling constant should be 3.0 to 3.5 instead of 4.5 to 5.0. This can be clearly seen in the case of deoxy- adenosine [16,17].

Free rotation around the Cl‘-C‘2 linkage and consequently also around the C3‘-C4‘ linkage is only possible if the original plane of Cl’, C2‘, C3’ and

Vol. 24, No. 2,1971

100

ki c

50

20

10

F. VON DER HAAR, E. SOE~LIMME, M. G~MEZ-GUILLEN, and F. CRAMER 301

OO 10 20 30 40 50 6'0 O O W / so . sb ' l o o ' 120 Time (min) Time (min)

E'ig.3. Pyrophaphmolysis of ( A ) yeast tRNAPhe-C-C-["H]A (0 ) and tRNAPhe-C-C-['H]Aoxr.red ( X ) and (B) yeast tRNAPhe- [14C]C-[l4C]C (0 ) and tRNAPhe-[14C]C-[14C] Coz'-red ( X) with tRNA-nucleotidyl transferase. The sample (100 pl) contained

0.3 A,,, unit tRNA. tRNA : enzyme ratio 1 A,,, unit: 165 pg. 10 pl samples were removed; A, blank without enzyme

Table 3. Pyrophospholysis of yeaat tRNAPhe with tRNA-nucleotidyl transferaae The amount of released C,, waa calculated as in Table 2

Substrate (0.06 Arw unit/100 vl) Enzyme

Intact 3'4erminus after

6 min 120 min Monitored base

vg/A,.. unit tRNA "0 % 42 7.5

165 7.5 165 9.0

'76

4 . 5

74 75 43 6 54 5

165 7.5 330 7.5

~~~ ~

60 31 51 23

tRNAPhe-[14C]C-[14C]C 160 7.5 58 14

160 7.5 99 - 77 88a

a The velocity-determining step is the hydrolysis of c 7 S &red. In order to calculate the value of removal of C7s Ofi-md, half the amount of radio- activity released has to be taken.

Table 4. Proton-magnetic-resonance data of AMPoxi-red and ATPoxr-red Chemical shifts are given as T values (ppm); internal standard was trimethylsilylpropane

= multiplet; the number of protons is given in parentheses. The coupling constants, J l / , %!. sulfonic acid. The multiplicity is given by: s = singlet, d = doublet, t = triplet, m .....

are given in Hz

H, H. H; H l El, H/ and H/

Hz HZ

m o x i - r e d 1.79 6 (1) 1.59s (1) 3.95t (1) 5 5.89 d (2) 5 6.15m (5) ATPoxi-rea 1.77 s (1) 1.55s (1) 3.94 t (1) 4.5 5.9Od (2) 4.5 6.12 m (5)

302 F. VON DER HAAR et al.: Substrate Properties of Modified Yeast tRNAPhe Eur. J. Biochem.

C4’ is distorted by rotation around the Cl’-O-C4’ acetal linkage. This can be demonstrated by space- filling models.

DISCUSSION

Oxidation with periodate and reduction with borohydride can be carried out on the monomer level as well as on the polymer level leading to a molecule with a ring-opened terminal ribose, e.g. ATPoxi-red from ATP and tRNAPhe-C-C-Aox~.red from tRNAPhe-C-C-A. The proton-magnetic-reso- nance data of ATPoxi-red indicate that the original plane of the ribose is distorted by rotation around the Cl’-O-C4’ linkage (Table 4). The thus modified tRNA, tRNAPhe-C-C-Ao,i-red, can still be aminoacylated [l]. According to Zachau et al. [18,19] the 3‘-hydroxy group is aminoacylated and therefore this group is still susceptible in the modified product, indicating that its stereochemistry is not greatly altered. On the other hand, aminoacylation of tRNAPhe-C-C-A in borate buffer is significantly slower than in Tris buffer (Fig. l), whereas no such decrease is observed with tRNAPhe-C-C-Aoxi.,ed (Fig. 1, Table l), suggest- ing that the 2‘- and 3’-hydroxy groups no longer form a borate complex of the same stability, presumably because of distortion of the hydroxy groups. Phe-tRNAP”’-C-C-Aoxi.red shows a 2.5-fold increase in half-life in Tris buffer pH 7.5 compared to Phe-tRNAPhe-C-C-A, indicating that the hydro- lytic lability of the aminoacyl group in aminoacyl- tRNA is largely due to the cis-glycol group.

tRNAPhe-C,,-C,,-A,, oxj-red is still degraded by snake-venom phosphodiesterase with a slight decrease of rate. The equivalent modification a t C;,, tRNAPhe- C,,-C,, oxi-red results in a molecule nearly completely stable towards attack of this enzyme (Fig.2, Table 2).

A similar distinction with respect to this equi- valent modification in A,, and C,, is found in the pyrophosphorolysis with tRNA nucleotidyl trans- ferase. Modification a t A,, has no significant influence, while modification a t C,, reduces the rate of enzy- matic attack to almost zero (Fig.3, Table 3).

Apparently in tRNAPhe the 3’-terminal A,, oxi-red has more resemblance to a ribose conformation than the 3‘-terminal C,, oxi.red. On the other hand ATPoxi-red is not an ATP analog in nucleotidyl transfer, and from the proton-magnetic-resonance data we know its non-ribose-like conformation. If, therefore, this ribose-like conformation is a reguire- ment for these enzymatic degradations, such a con- formation must be generated either by involving the 3‘-terminal A,, oxi-red in the three-dimensional struc- ture of tRNA or during the enzymatic reaction. It has already been shown by physicochemical measure- ments [20-22] that the 3’-terminal A is involved in the overall structure of tRNA.

For skilful technical assistance we are greatly indebted to Miss D o h Niehus and Miss Renate Waldmann. tRNA nucleotidyl transferaae waa prepared by Miss Erika Gaert- ner. The proton-magnetic-resonance spectra were determined by Dr M. Schiebel (Gesellschaft fur nwlekularbiologische Forschung, Braunachweig-St6ckheim). We are grateful to Dr D. Gauss and Dr M. Sprinzl for critical discussions and helpful suggestions. One of us (M.G.-G.) is very indebted to the Max-Planck-Gesellschaft for obtaining a special fellow- ship.

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F. von der Haar, E. Schlimme, and F. Cramer* Max-Planck-Institut fur Experimentelle Medizin Abteilung Chemie BRD-3400 Gottingen, Hermann-Rein-StraBe 3 German Federal Republic

M. G6mez-Guillen’s present address: Departamento de Quimica Orgknica, Facultad de Ciencias Universidad de Sevilla, Sevilla, Spain

* To whom requests for reprints should be sent.