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Action of venom phosphodiesterase on transfer RNA from Escherichia coli
176
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96590
ACTION OF VENOM P H O S P H O D I E S T E R A S E ON T R A N S F E R RNA FROM E S C H E R I C H I A
j
COLI
p. M I L L E R , M. E. H I R S T - B R U N S AND G. R
PHILIPPS
Department o[ B*ochemzstry, St L o i s Unzverszly School of Medicine, St. Lou*s, Mo. 631o 4 (U.S.A ) (Received March 23rd , 197 o)
SUMMARY
Digestion of t R N A by venom phosphodlesterase was assayed under a variety of conditions. Released nucleotldes were characterized and the amount determined b y chromatography on Dowex I-X8 The release was also determined b y readdltlon of AMP and CMP to digested t R N A usmg purified tRNA-nucleotmdyltransferase. The two methods gave identical results as long as the fourth nucleotlde from the 3'-OH terminus was not removed. The rate of removal of each of the three terminal nucleotides was determmed. The terminal AMP was removed about 4 times faster than the first CMP and 65 times faster than the second CMP. At 37 ° the rate was 3-5 tnnes faster than at 20 ° for all three nucleotides. No sigmficant differences were seen for a n y particular ammo acid-specific tRNA. The presence of an amlnoacyl group at the 3'(2') terminus of t R N A decreased the rate of hydrolysis of the terminal AMP by a factor of 5. Differences of the release of aminoacyl-O-AMP were seen for five different a m i n o a c y l - t R N A ' s tested.
INTRODUCTION
t R N A can serve as substrate for tRNA-nucleotidyltransferase (EC 2.7.7.25) after partial or complete removal of the 3'-terminal trinucleotide. This can be accomplished by limited digestion with venom phosphodiesterase (orthophosphorlc diester phosphohydrolase, EC 2.1. 4 I) from Crotalus adamanteus 1,2. The enzyme attacks t R N A from the 3'-OH end and consecutively hberates 5'-mononucleotides~,4. ZUBAY AND TAKANAMI5 a n d ROSSET et al 6 reported t h a t at 20 °, venom phosphodiesterase liberates only about I mole of AMP and 1.2 moles of CMP per mole of tRNA. At 37 ° degradation proceeds further. Under suitable conditions, the complete 3'-OH terminal trinucleotlde sequence can be removed a n d degradation continues beyond this point at a reduced rate 1,8,4,7. Here we report the results of a systematic study of the controlled digestion of Escherichm coh t R N A by venom phosphodmsterase. The rate of venom phosphodiesterase-medlated removal of each of the three terminal nucleotldes was determined at 20 and 37 °. We therefore conclude t h a t no conformatlonal change near the 3'-OH end of t R N A occurs between 20 and 37 °. We also compared the rate of hydrolysis of the terminal adenosyl residue of Bzochz~n Bzophys Acta, 217 (197 ° ) 176-188
ACTION OF VENOM PHOSPHODIESTERASEON tRNA
177
aminoacylated and deacylated tRNA. It had been reported s that the presence of an aminoacyl group in the 3'(2') position of the terminal adenosine of t R N A does not affect the rate of hydrolysis of tRNA by venom phosphodlesterase. Under the more stringent conditions used here differences in rate of the removal were observed.
MATERIALSAND METHODS 3H-labeled triphosphate nucleosides and E14C]amlno acids were obtained from Schwarz BioResearch, Inc., Orangeburg, N. Y. ATP, CTP, and Dowex I-X8 were from Calblochem, Los Angeles, Calif. Bis-p-nitrophenyl phosphate was from Sigma Chemical Company, St. Louis, Mo. Glass fiber filters 934 AH were from Reeve Angel, Clifton, N. J. Protein was determined by the method of LOWRY et al. 9 using bovine selum albumin as standard. For the estimation of t R N A concentration, an average molecular weight of 26 ooo and an absorbance in 0.2 M NaCl of A1% "-,,60 am : 220 was used 1°.
Enzymes and R N A Venom phosphodlesterase (Lot No. 8KA) was purchased from Worthington Biochemical Corporation, Freehold, N. J. It was dissolved in IO mM magnesium acetate, IO ~o glycerol to give a I.O mg/ml solution and was stored at --20 °. No loss of enzymic activity was observed over a period of 4 months, tRNA-nucleotidyltransferase was purified as previously described 11. Aminoacyl-tRNA synthetases were prepared by a modification of the method of MUENCH AND BERG12. Cells from E. col~ Strain A-I 9 were used and ribosomes and t R N A removed by liquid phase polymer fractionation ~l. The lower phase obtained after (NHa)2SO 4 precipitation was dialyzed against phosphate buffer and chromatographed on DEAE-cellulose 1~. This synthetase preparation was free of tRNA-nucleotidyltransferase, ribonuclease I, and tRNA. t R N A was purchased from Schwarz BioResearch and purified on Sephadex G-Ioo (ref. 13) Purification of tRNA val will be described elsewhere. Venom phosphodiesterase was assayed by a modification of the method of KOERNER AND SINSHEIMER 14. The increase with time of Aa0o nm was determined for a o.52-ml reaction mixture containing I I #moles bis-p-nitrophenyl phosphate, 21/,moles glycme-NaOH (pH 8 7), 6 #moles magnesium acetate, and lO'-2o #g venom phosphodiesterase. One enzyme unit was defined as that amount of protein which liberated one nmole p-mtrophenol per rain at 25 °.
Venom phosphodiesterase digestzon o / t R N A and chromatographic analys~s o/nucleot,des The Incubation mixture contained in 0.5 ml: 20 #moles glycine-NaOH (pH 8.7), 5 #moles magnesium acetate, 147 nmoles tRNA, and 5 o - 5 o o # g venom phosphodiesterase (147 unlts/mg). After incubation for different times, the mixture was apphed to a 0. 9 cm ×5o cm column of Sephadex G-25 with a layer of 0.2 g acidwashed sllicic acid 15 (IOO mesh) on top. Immediately before the sample was applied, 5 ml of IO mM Tris-HC1 (pH 7-3), I mM magnesium acetate, 300 mM NaC1 was run onto the column. The column was then eluted with o.I M sodmm acetate (pH 4-4) at 0.2 ml/min. After the t R N A peak had been eluted, the outlet of the Sephadex column was connected to the inlet of a I cm × 9 cm Dowex I-X8 (400 mesh) column
B*och*m B~ophys. Acta, 217 (197o) 176-188
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j . P . MILLER et al.
equilibrated with the same buffer. The low-molecular-weight peak from the Sephadex column was thus quantitatively transferred to the Dowex column. The Dowex column was washed with IO ml of o i M sodium acetate (pH 4.4) and then eluted with a linear gradient of 5 ° m l o.I M sodium acetate (pH 4.4) and 50 ml 3.o M sodium acetate (pH 4.4) The A2¢0 nm of the eluate was monitored and recorded. The elution volume and the area under each peak were determined for each nucleotlde. They were identified by their A2s0 nm/A260 nm ratio. A mixture of known amounts of the major nucleotides and adenosine was chromotographed on an identical column. Recovery was greater than 95 %- The nucleotldes released after venom phosphodlesterase digestion were quantltated b y direct spectrophotometrlc analysis or by comparison of the area under each peak with that of the standard column The two methods gave essentially the same results. The t R N A from the Sephadex column was used in the assays described below
Enzymatic add#,on o/nucleotides to venom phosphodiesterase d,gested t R N A Extent of t R N A digestion was also determined by enzymatic readdltlon of nucleotldes. Fractions from the Sephadex column containing t R N A were pooled and precipitated with ethanol The t R N A was collected by centrlfugation and the pellet dissolved in I mM Tns-HC1 (pH 7-5), IO mM magnesium acetate. The assay mixture contained in o.I ml: 5/,moles glycine-NaOH (pH 9.2), I / , m o l e magnesium acetate, I/~mole GSH, 0.2-0.6 nmole venom phosphodlesterase-treated tRNA, 20 #g bovine serum albumin, and I 4-4.2 units (1-3/zg) tRNA-nucleotldyltransferase. For AMP incorporation the mixture also contained lO.4 nmoles (12.4 nC) of [8-3HIATP and 5 6 nmoles CTP; for CMP incorporation it contained 15. 4 nmoles (23.2 nC) of [5-aHICTP either in the presence or absence of 60.0 nmoles ATP. For the incorporation of GMP and UMP 19.6 nmoles (18. 7 nC) E3HIGTP or 16.1 nmoles (15.9 nC) ~5-3HIUTP were used. All assays were done in duphcate. Incubation was for I h at 37 ° The reaction was stopped b y the addition of 2 ml of ice-cold 20 mM EDTA, 0.2 mM ATP and 2 ml of IO % trichloroacetlc acid. The precipitate was collected on glass fiber filters. Tubes and filters were washed twice with 3.5 % trlchloroacetic acid and the filters once with 95 % ethanol. They were dried and counted in 3.0 ml of a toluenebased scintillator in a Packard 3380 liquid scintillation spectrometer. To produce larger amounts of labeled tRNA, 14o nmoles venom phosphodiesterase-treated t R N A and IOO units tRNA-nucleotidyltransferase were incubated in 2 ml of the same buffer. Either 2.48/,moles (2.95 pC) ~8-3HlATP and 0.63/~moles CTP or o.7o/~moles (1.o5/~C) [5-aHICTP and 2.80 /,moles ATP were used After Incubation for I h at 37 °, the mixture was applied to a Sephadex G-25 column equilibrated with IO mM Trls-HC1 (pH 7.3), I mM magnesium acetate, 0.3 M NaC1; with 0.5 g acid-washed silicic acid layered on top. Fractions containing t R N A were pooled precipitated with ethanol, and the precipitate dissolved in I ml of I mM Tris-HC1 (pH 7 5), IO mM magnesium acetate Determination o/amino acid acceptor act,wty A typical reaction mixture contained in 7 ° # 1 : 7 5 / , m o l e s sodium cacodylate (pH 7.2), 7.5/,moles of KC1, 1.2/~moles of magnesium acetate, o 175/~mole ATP, 0.725/zg bovine serum albumin, 16o pmoles of tRNA, 9/~g crude ammoacyl-tRNA synthetases, 80 pmoles of [14Clamlno acid, and 6.4 nmoles of the other nineteen Bzoch,m Bzophys. Acta, 217 (197o) 176-188
ACTION OF VENOM PHOSPHODIESTERASE ON t R N A
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a m i n o acids. Th e r e a c t i o n m i x t u r e was i n c u b a t e d for IO m i n at 37 °, s t o p p e d b y ad d i tion of 0.5 ml of 7 ~o trichloroacetic acid c o n t a i n i n g an excess of u n l ab el ed a m i n o acids, a n d t h e p r e c i p i t a t e d t R N A collected on glass fiber filters. Th e filters were washed w i t h trlchloroacetic acid a n d ethanol, dried an d c o u n t e d in 3 ml of a toluenebased scintillator.
RESULTS
E]/ect o/ temperature and time on venom phosphodiesterase digest*on T h e e x t e n t of digestion of t R N A b y v e n o m phosphodiesterase was assayed b y analysis of the nucleotides released. N u c l e o ti d e s a n d t R N A were s e p a r a t e d on Sephade x G-25 an d t h e nucleotides s e p a r a t e d on D o w e x I - X 8 . Fig. I shows the results of a 2o ° digestion. Th e a m o u n t of A M P released from t R N A increased r a p i d l y a n d reached a m a x i m u m after I h. CMP was r e m o v e d at a n o t i c e a b l y slower rate. T h e increase in CMP r e m o v a l after 4 h p r o b a b l y r ep r esen t ed release of t h e t h i r d nucleotide f r o m t R N A . A small a m o u n t of U M P was also d et ect ed , while no G M P was released. No adenosine was f o u n d in t h e low-molecular-weight fraction f r o m the S e p h a d e x column. This suggested t h a t 5'-nucleotidase (EC 3.1.3.5) a n d rlbonuclease a c t i v i t y (EC 2.7.7.17) were absent. T h e t R N A recovered f r o m S e p h a d e x was st u d i ed for its ab i l i t y to i n c o r p o r a t e nucleotides e n z y m a t i c a l l y . The a m o u n t of A M P r e m o v e d b y v e n o m phosphodiester i s e e q u a l e d the a m o u n t t h a t could be a d d e d back. I n Fig. 2 t h e results of the enzy-
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Fig. I. Digestion of tRNA by venom phosphodmsterase at 20 ° Dlgestmn was performedIas described in MATERIALSAND METHODS using 14 7 unlts/ml venom phosphodiesterase and 294 nmoles/ml t:RNA. Nucleotldes released were assayed by chromatography on Dowex I-X8. × - ×, AMP removed, {} - 0 , CMP removed, O - Q , UMP removed Fig. 2. Addition of CMP by tRNA-nucleottdyltransferase to tRNA digested at 20 ° The tRNA was the same as used m F]g. i Nucleotlde addition was performed as described in MATERIALSAND METHODS using 2.8 units tRNA-nucleotldyltransferase and o 3 nmole tRNA per assay. { } - 0 , CMP loss as assayed by Dowex I-X8 chromatography (from Fig I), O- - -O, CMP addition an the presence of o.6o mM ATP; × - x , CMP addition m the absence of ATP.
B,och,m. Bwphys Acta, 217 (I97 o) i76-188
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matic a d d i t i o n of CMP are shown. W h e n assayed in the absence of A T P a p p r o x i m a tely twice as m u c h CMP was a d d e d to t R N A as was released. This m a y be caused b y the f o r m a t i o n of t R N A - p C p C p C , a possibility which is c u r r e n t l y u n d e r investigation. W h e n a d d i t i o n was studied in the presence of 0.6 mM ATP, a d d i t i o n of CMP equaled the release. I n c o r p o r a t i o n of U M P was 0.2 mole/mole t R N A m all instances
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Fig. 3 Digestion of tRNA by venom phosphodlesterase at 37° The conditions were the same as m Fig I x - x , AMP removed, O - O , CMP removed, Q - O , GMP removed, O - - - 0 , UMP removed Fig 4 Addition of AMP by tRNA nucleotldyltransferase to tRNA digested at 37°. The tRNA used was the same as in Fig 3, 4 2 umts tRNA-nucleotldyltransferase and o 5 nmole tRNA were used per assay 0 - 0 , AMP loss as assayed by Dowex I-X8 (from Fig. 3), C) O, AMP addition. Fig. 3 shows the results of v e n o m phosphodlesterase digestion at 37 °. At the shortest time investigated, i e. after 30 rain, I mole of AMP a n d 1. 4 moles of CMP per mole of t R N A h a d been released. Thereafter, the release of CMP increased linearly up to a p o i n t where 2.2 moles of CMP h a d been removed. On the other h a n d , the a m o u n t of AMP In the nucleotide fraction r e m a i n e d c o n s t a n t for 4 h. Since AMP is f o u n d in the fourth position from the 3'-OH t e r m i n u s i n 69 ~o of E. coh t R N A a6, the increase after 4 h p r o b a b l y represents r e m o v a l of the fourth nucleotide. The time p o i n t where more t h a n I mole of AMP was released coincided with t h a t where 2 moles CMP were released. Both U M P a n d GMP appeared in the m o n o n u c l e o t l d e fraction from t R N A digested b y v e n o m phosphodiesterase at 37 °. I n Fig 4, the e n z y m a t i c r e a d d l t l o n of AMP was investigated. I mole of AMP was a d d e d to all t R N A digested for u p to 4 h. t R N A which h a d been digested longer accepted less AMP t h a n h a d been removed. Fig 5 shows a d d i t i o n of CMP to the same t R N A I n the absence of A T P a n extraneous a d d i t i o n of CMP was again seen, howBzochzm Bzophys Acta, 217 (197° ) I76-I88
ACTION OF VENOM PHOSPHODIESTERASE ON
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F i g 5 A d d i t i o n of C M P b y t R N A - n u c l e o t l d y l t r a n s f e r a s e t o t R N A d i g e s t e d a t 37 °. t R N A w a s t h e s a m e as t h a t used in Fig 3 O - Q , CMP loss as a s s a y e d b y c h r o m a t o g r a p h y on D o w e x I - X 8 (from Fig 3), C) - - - O , CMP a d d i t i o n in t h e p r e s e n c e of o 6o mM A T P , × - × , C MP a d d i t i o n in t h e ab sen ce of AT P.
ever the amount of CMP added above that which had been removed was only o.4 mole/mole tRNA. When CMP addition was assayed in the presence of ATP, incorporation equaled removal for up to 4 h. After that the abihty of t R N A to incorporate CMP decreased rapidly. Addition of UMP and GMP to tRNA digested at 37 ° was also investigated. No GMP could be incorporated regardless of the extent of digestion. Incorporation of UMP to tRNA gave values of approx. 0.25 mole/mole t R N A when digestion was up to 4 h; thereafter the values were slightly higher.
E//ect o/venom phosphodiesterase concentration on the extent o / t R N A hydrolysis The data of the preceding experiments suggested that the removal of the third nucleotide was very slow. To investigate this further, the removal of the third nucleotide was studied with higher venom phosphodiesterase concentrations. As shown in Expt. A of Table I, higher venom phosphodiesterase concentrations resulted in the removal of the third nucleotide, while the amount of AMP released remamed constant. All AMP and CMP residues which had been removed could be enzymatically reincorporated This shows that the complete -pCpCpA sequence can be removed at 20 ° but that larger amounts of enzyme and longer incubations are needed. At the highest enzyme concentration, incorporation of nucleotides into tRNA was less than the amount released. Comparing the AMP removal with the addition it is suggested that at least 5 % of all tRNA molecules had been digested beyond the third nucleotide. The addition of CMP lndmated that the third nucleotlde had been hberated from only about 80 % of all t R N A molecules While the amount of GMP released was proportional B,och*m. B~ophys. Acta, 217 (197 o) I 7 6 - I 8 8
J . p . MILLER et al.
182 TABLE
I
EFFECT OF VENOM PHOSPHODIESTERASE CONCENTRATION ON THE EXTENT OF DIGESTION OF t R N A t R N A w a s digested w i t h v e n o m p h o s p h o d l e s t e r a s e for 6 h a n d the nucleotldes r e m o v e d a n a l y z e d a s d e s c r i b e d u n d e r MATERIALS AND METHODS. T h e a s s a y f o r r e a d d l t l o n of A M P , G M P , C M P a n d U M P , u s i n g 2 8 u n i t s of t R N A n u c l e o t l d y l t r a n s f e r a s e a n d 0.2 n m o l e t R N A w a s p e r f o r m e d a s d e s c r i b e d in MATERIALS AND METHODS C M P i n c o r p o r a t i o n w a s d e t e r m i n e d in t h e p r e s e n c e of o 6 mM ATP.
Expt.
Temp
Enzyme conch (unzts/ml)
A
203
14. 7 44.1 73.5 148
B
37 °
147 44 I 735
Nucleotzdes removed (moles~mole t R N A )
Nucleot~des added (moles~mole t R N A )
AMP
CMP GMP
UMP AMP
C M P G3/IP U 3 I P
I I I I
I I I I
o.98 o 97 I GO o 94
1.o 7 I o7 1.46 I 78
o o o o
o89 o29 ooo
I97 o88 ooo
ooo ooo ooo
GO o3 07 22
I 08 I 96 271
05 19 36 91
o GO o.II o 19 0.72
o o o o
19 34 57 69
226 249 269
o79 I 43 2o 5
o69 I 15 I 59
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o o o o
20 21 27 28
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to the amount of enzyme, none could be reincorporated. Likewise, UMP was released in increasing amounts but remcorporation remained the same for all samples. In Expt. B of Table I, the results of a digestion at 37 ° are shown. At the lowest enzyme concentration all t R N A had lost the -pCpCpA terminus and IO % had lost four nucleotmdes. At an enzyme concentration of 44.1 umts/ml digestion had proceeded beyond the third nucleotide in 60-70 % as determined by CMP incorporation. The data suggested that approx, o. 7 mole of AMP was released from TABLE
I1
COMPARISON OF RATES ON VENOM PHOSPHODIESTERASI~ DIGESTION FOR EACH OF THE THREE TERMINAL NUCLEOTIDES OF t R N A
T h e t R N A - p C p C p [ S H ] A w a s p r e p a r e d b y a d d i t i o n of C M P a n d ESH]AMP t o i R M A - p C , t R N A pCp[3H]C was made by reconstituting iRMA-pC with [3H]CTP and ATP and redlgestmg the r e s u l t i n g t R N A t o r e m o v e m o s t of t h e t e r m i n a l A M P . T h i s t R N A c o n t a i n e d a p p r o x 45 % t R N A - p C p [ s H I C (15 % t R N A - p C p [ S H I C p A a n d 4 ° % i R M A - p C ) T h e t R N A - p E S H ] C w a s m a d e by reconstituting tRNA without CpCpA with [~H]CTP and ATP, the termmal dlnucleotlde w a s t h e n r e m o v e d . I n c u b a t i o n w a s a s d e s c r i b e d e x c e p t t h a t 2 o / 2 m o l e s of t R N A w e r e u s e d A h q u o t s of IO/21 w e r e r e m o v e d a n d p r e c i p i t a t e d w i t h t r l c h l o r o a c e t l e a c i d T h e a s s a y f o r b~s-pm t r o p h e n y l p h o s p h a t e w a s d e s c r i b e d i n MATERIALS AND METHODS
Substrate
Enzyme (t~g/ml)
Nucleotzderelease (pmoles/m*n p e r ~ g venom phosphod*esterase) 20 ° 37 °
Increase ~n rate at 37 ° over 2o ~ (-[old)
t R N A - p C p C p L3H7A
IO 3 2o 5 14 6 29 2 29.2 584
21 20 5 4 o o
3 3 4 4 5 5
tRNA-pCp[sHJC t R N A - p [~H1C B1s-p-mtrophenyl phosphate
38 5
8 o 54 59 25o 233
5 7 ° . lO4
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71.o 62 8 24 9 22.5 I 29 i 19 297
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the fourth position 16 while the remaining o 3 mole came from positions beyond the fourth nucleotide. To completely remove the fourth nucleotide, an enzyme concentration of 73-5 umts/ml had to be used. In this case, an average of 9 moles of nucleotides were removed. Therefore it might be assumed t h a t after the terminal trinucleotide was released t R N A was hydrolyzed at a faster rate. Release of UMP and GMP at 37 ° was also investigated. The amount increased with increasing enzyme concentrations When the fourth nucleotide was completely removed, 2.05 moles GMP and 1.59 moles UMP had been liberated. The amount of GMP and UMP released with 14. 7 umts/ml enzyme at 37 ° and 148 units/ml enzyme at 20 ° was the same Since under these conditions the amounts of AMP and CMP removed a p p r o x i m a t e l y equaled t h a t which could be added back, it appears t h a t GMP and UMP came from positions in t R N A t h a t do not effect the readition of the -pCpCpA terminus.
Rate o/removal o/term,nal nucleotides The results described so far suggested t h a t the three terminal nucleotides were removed from t R N A at different rates. In order to s t u d y these differences in greater detail, tRNA-pCpCpE3HIA, tRNA-pCpE3HIC, and tRNA-p[~HIC were prepared. The method for preparation is described in Table II. The rate of loss of 3H-labeled nucleotides was determined at 20 and 37 °. Two different enzyme concentrations were used and the rate was proportional to the enzyme concentration in each case. The terminal AMP was removed 3-4 times faster than the first CMP, and 50-80 times faster t h a n the second CMP. The increase in rate at 37 over 20 ° was 3-5-fold for all substrates. This showed t h a t the p r i m a r y difference between 20 and 37 ° is only a difference in rate and suggested t h a t the increase in velocity at 37 ° could be t o t a l l y accounted for b y an acceleration of the enzymatic catalysis. TABLE
III
AMINO ACID ACCEPTANCE OF RECONSTITUTED t R N A t R N A w a s d i g e s t e d 4 h a t 2 0 ° t o r e m o v e o n e A M P a n d t w o C M P r e s i d u e s ( C o l u m n 2) a n d d i g e s t e d 4 h a t 37 ° t o r e m o v e o n e A M P a n d t w o C M P r e s i d u e s B o t h t R N A ' s w e r e t h e n r e c o n s t i t u t e d . For amlnoacylatIon an enzyme preparation free of tRNA-nucleotldyltransferase was used.Inc u b a t i o n w a s f o r i o m m a t 37 °. T h e c o n t r o l t R N A w a s f r o m t h e s a m e l o t b u t u n t r e a t e d The asterisk indicates nucleotldes removed and added back
A ~nz~lo acid
Ala Arg Asp Glu Gly His Ile Leu Lys Met Phe Pro Set Tyr Val
A m , n o ac,d *ncorpor at*on (pmoles ) Control
tnXA-pCpC'pA"
tnNA@C'pC'pA"
47 35 12 18 7 19 16 87 23 37 25 12 25 22 55
49 26 16 II 4 19 17 46 17 36 27 15 14 15 42
5° 33 13 13 7 24 8 77 25 24 25 15 21 12 31
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Amino ac,d acceptance o/ reconstituted t R N A To d e t e r m i n e w h e t h e r v e n o m p h o s p h o d l e s t e r a s e a t t a c k s all a m i n o acid accepting t R N A species, t R N A was digested w i t h v e n o m p h o s p h o d i e s t e r a s e to remove increasing a m o u n t s of t h e t e r m i n a l d m u c l e o t i d e . F o r t e n a m i n o acids tested, we o b s e r v e d t h a t the loss of a m i n o acid a c c e p t a n c e increased w i t h t h e progress of v e n o m p h o s p h o d l e s t e r a s e digestion. There were no gross differences b e t w e e n different a m i n o acids A m i n o acid a c c e p t o r a c t i v i t y of d i g e s t e d t R N A after r e c o n s t l t u t l o n was also t e s t e d (Table I I I ) . The a c c e p t o r a c t i v i t y was d e t e r m i n e d for t R N A d i g e s t e d at 20 ° to r e m o v e one A M P a n d CMP a n d a t 37 ° to r e m o v e one A M P a n d two CMP residues. B o t h t R N A were t h e n r e c o n s t i t u t e d a n d t h e a m i n o acid a c c e p t a n c e c o m p a r e d w i t h t h a t of an u n t r e a t e d s a m p l e (Column I). No significant loss of acceptor a c t i v i t y was observed w i t h respect to a n y a m i n o acid. These d a t a suggest t h a t no i n d i v i d u a l species of t R N A was p r e f e r e n t i a l l y d i g e s t e d b e y o n d the t h i r d nucleotlde a n d t h a t t R N A - n u c l e o t i d y l t r a n s f e r a s e reacts w i t h all a m i n o acid-speclhc t R N A specl,.S
Venom phosphodiesterase dzgest,on o/ ammoacylated tRNA I n t h e course of these studies we a t t e m p t e d to p r o t e c t b y a m i n o a c y l a t i o n md l v i d u a l a m i n o acid-specific species from e n z y m a t i c a t t a c k b y v e n o m p h o s p h o d l esterase. Since a m i n o a c y l a t i o n d i d not p r o t e c t t R N A from e n z y m a t i c digestion, the effect of v e n o m p h o s p h o d l e s t e r a s e on a m i n o a c y l - t R N A was i n v e s t i g a t e d m more detail, t R N A was labeled w i t h aH in the 3 ' - A M P residue a n d one aliquot was t h e n a m i n o a c y l a t e d . Using the same c o n c e n t r a t i o n of b o t h s u b s t r a t e s the loss of trichloroacetlc acid-precip]table r a d i o a c t i v i t y as [ a l l ] A M P or a m i n o a c y l - I S H ] A M P was d e t e r m i n e d (Fig. 6A). To a v o i d s p o n t a n e o u s h y d r o l y s i s of the a m i n o acid ester b o n d i n c u b a t i o n was done at 23 ° a n d p H 7 2 The e n z y m e c o n c e n t r a t i o n was higher in this
I00
I00
i
i
,
i
,
r
i
i
i
i
i
i
i
i
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80
o~ 80
60
z_ 60
_z
~ 4o
4o
t~
b_ 20 J I
P
2
I
I
I
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4 6 TIME (MINI
I0
20 30 TIME (MINI
40
Fig. 6 A Effect of v e n o m p h o s p h o d l e s t e r a s e on t h e release of E3HIAMP from d e a c y l a t e d a n d a m m o a c y l a t e d t R N A The c o n c e n t r a t i o n s were p e r ml 8 6 nnl ol e s d e a c y l a t e d tRlXlA or 8 o n m o l e s a m m o a c y l a t e d t R N A , 143/,g v e n o m p h o s p h o d l e s t e r a s e m 5 n m o l e s Trls-HC1 (pFI 7 2), i o nmol e s Mg a c e t a t e A h q u o t s of a p p r o x , i o o p m o l e s t R N A were r e m o v e d a t i n t e r v a l s of I mln, s p o t t e d ont o M l l h p o r e h l t e r s , s o a k e d in IO % t r l c h l o r o a c e t l c acid, dri e d a n d c o u n t e d The specific a c t i v i t y of t h e 8H-labeled t R N A w a s a b o u t 31oo c o u n t s / r a m per nmole. Q - Q , a mmoa c yl -t R 1Y A , O - O , d e a c y l a t e d t R N A B. E f f e c t of v e n o m p h o s p h o d l e s t e r a s e on t h e release of [3H]CMP from d e a c y l a t e d a n d a m m o a c y l a t e d t R N A The a s s a y w a s p e r f o r m e d as d e s c r i b e d for A e x c e p t t h a t t he t R N A w a s l a b e l e d w i t h [3H]CTP 18 n m o l e s / m l of d e a c y l a t e d or 14 n m o l e s / m l a m l n o a c y l a t e d tRlXIA were i n c u b a t e d w i t h 1 4 3 / , g / m l of v e n o m p h o s p h o d l e s t e r a s e I n c u b a t i o n w a s c a rri e d o u t for a t o t a l of 4 ° m m a n d a h q u o t s were r e m o v e d e v e r y 5 m m The specific a c t l v l t y of t h e 3H-labeled t R N A w a s a b o u t i 6 o o c o u n t s / r a m p e r n m o l e (7)-(7), a m m o a c y l - t R N A ; O - Q , d e a c y l a t e d t R N A .
B*och*m B~ophys. Mcla, 217 (197 o) 176-188
A C T I O N OF V E N O M P H O S P H O D I E S T E R A S E
185
ON tRNA
experiment in order to observe AMP release over a shorter time period. The initial rate for deacylated t R N A was about 5 times the rate for amlnoacylated t R N A . In Fig. 6B removal of the adjacent CMP by venom phosphodmsterase was compared for deacylated and aminoacylated tRNA. The assay conditions were the same except that the incubation was for 4 ° rain, thus the slope only appears steeper in Fig. 6B. Again, the rate of digestion was 4 times faster for deacylated tRNA. At I rain, 3 1 % of the terminal AMP and about 8 % of the following CMP were removed from deacylated t R N A while these values were 6 and 2 %, respectively, for aminoacylated tRNA. Thus, the ratio of the initial rate for deacylated v s . aminoacylated tRNA was approximately the same in both cases. This indicated that the presence of the amino acid group retarded the removal of the 3'-terminal AMP residue only. TABLE
IV
THE EFFECT OF VENOM PHOSPHODIESTERASE ON AMINOACYL-tI{k'~A t R N A w a s a m l n o a c y l a t e d a n d p u r i f i e d a s d e s c r i b e d m MATERIALS AND METHODS 16 n m o l e s o f t h e v a r i o u s [ 1 4 C ] a m l n o a c y l - t R N A ' s w e r e i n c u b a t e d i n i m l w i t h i o / z m o l e s T r l s - H C 1 ( p H 7 4), i o p m o l e s m a g n e s i u m a c e t a t e , 4 ° / * m o l e s KC1 w i t h o r w i t h o u t 2 0 p g of v e n o m p h o s p h o d l e s t e r a s e a t 37 °. T h e r e a c t i o n w a s s t o p p e d b y a d d i t i o n of I m l o f i o % t r l c h l o r o a c e t l c a c i d a n d t h e p r e c i p i t a t e collected on Mllllpore filters The radmactivity determined as described
Venom Incubatzon Spee,#c radzoactw~ty phospho- T~me (counts/m~n per nmole t R N A ) d~esterase (re,n) Phe-tRNA Leu-tRN.4 Val-tRNA
--+ + +
o 2o o io 20
1°24 868 lOO2 608 349
5533 5o49 5489 4235 3171
H~s-tRNA
Am*noacyltRNA Ala-tRNA (algal protein hydrolysate )
574 541 569 415 331
1121 9oo lO42 794 732
57 ° 46o 57 ° 400 34 °
3754 2917 3159 2616 1337
ioo 94 99 72 57
ioo 80 93 71 65
ioo 81 ioo 7° 59
ioo 78 84 7° 36
Recovery of radzoactw~ O, (%) - -
- -
+ + +
o
20 o IO 2o
ioo 84 97 59 34
ioo 90 97 75 56
Digestion of deacylated tRNA by venom phosphodiesterase was about the same for all amino acid-specific species. To see whether the presence of a particular amino acid on t R N A affects venom phosphodiesterase digestion the experiment of Table IV was performed. Both spontaneous hydrolysis and the venom phosphodiesterasemediated release of labeled amino acids are shown. Release of the labeled amino acids from t R N A in the presence of venom phosphodiesterase differed for particular amino acids and these differences were unrelated to the variations observed for spontaneous hydrolysis.
DISCUSSION
The rates with which the 3'-terminal nucleotides of tRNA are enzymatically released by venom phosphodiesterase differ greatly. It is five times faster for the 3'B~och~m Bwphys. Acta, 2 1 7 (197 o) 1 7 6 - 1 8 8
186
j . p . MILLERet al.
terminal AMP than for the tollowing CMP residue and 8o times faster than for the next (second) CMP residue. This is not due to differences in the susceptlblhty to phosphodlesterase action among the four major nucleotlde bases 4. Due to the large difference m the rate of removal of the two CMP residues, only the terminal dlnucleotlde is liberated with low enzyme concentrations at 2o °. This confirms the results of others 1,~,5. At high concentrations of enzyme, the complete pCpCpA sequence call be removed at 20 °. Thus, our results exclude the hypothesis of ZUBAY AND TAKANAMI5 that the differences in the removal of the terminal nucleotldes between 20 and 37 ° are due to a weakening of the secondary structure of tRNA. This is substantiated by the observations that no gross changes exist in melting curves 17or optical rotatory dispersion curves is of t R N A in Mgz+ between 20 and 37 °. Therefore, the increase in rate of venom phosphodiesterase digestion between 20 and 37 ° is caused by a general effect of temperature on the reaction, rather than on the t R N A substrate. The t R N A used in these studies had been purified by chromatography on Sephadex G-Ioo (ref IOO). As we will describe elsewhere (G. R. PrlILIPPS, J. L. TIMKO AND T. W. MUNNS, in preparation), the t R N A was not contaminated by 5-S RNA, it gave a characteristic broad band after polyacrylamide gel electrophoresis. It was thus surprising to find a small amount of UMP in all digests at 20 °. ROSSET et al 6 made a similar observation but attributed this to a contamination with 5-S RNA: UMP is the 3'-termmal nucleotide of 5-S RNA of E. coh 19 Our results exclude this possibility. Also, UMP was incorporated mto tRNA while GMP was not. Incorporation of UMP into tRNA by rat liver tRNA-nucleotidyltransferase has been reported 20. FRESCO et al. 2~ suggested that the conformation of all tRNA species must be similar in order to fulfill the same functional requirements. That this might be indeed so, at least with respect to the -pCpCpA sequence, is suggested from our results which showed that no difference exists in rate or extent of digestion between different amino acid-specific tRNA's. Also, in prehminary experiments in which digestion of tRNA~~et and tRNAIval was studied, no gross differences were detected The reason for the different rates of removal of AMP and CMP may be the very structure of tRNA. All tRNA's of known sequence can be arranged in a cloverleaf conformation. Comparison of these structures 22 shows the fourth nucleotlde from the 3'-terminus is never hydrogen bonded. Thus the secondary structure cannot be the cause of the difference in rates of hydrolysis. The tertiary structure of tRNA, which is understood to be the folding of the different loops and the -pCpCpA stem into a more compact conformation, may be responsible for these differences. Various models have been proposed for the conformation of t R N A which differ with respect to the folding of the cloverleaf. LAKE AND BEEMAN 23 a n d CANTOR et al. 24 proposed an interaction between the anticodon loop and the -pCpCpA stem. DOCTORet al. 25, NINO et al. ~6, and LEVITT27 propose an interaction of the T~PGC loop and the amino acid stem. In the model of MELCHER 28, a hydrogen bond is formed between the 3'-terminal AMP residue of deacylated tRNA and the T 54 of the T ~ G C loop (in the nomenclature proposed by PHILIPPS 22) At the same time the AMP is positioned in close vicimty to the bases of the antlcodon The conformation proposed by CRAMERet al. zg, and its modification by M. STAEHELIN (personal communication) allows for an interaction of the -pCpCpA stem with the dlhydrouridine loop. This structure was originally proposed for yeast Bzoch~m Bzophys Acta, 217 (197° ) 176-188
ACTION OF VENOM PHOSPHODIESTERASE ON
tRNA
187
t R N A Phe but can be apphed to all t R N A molecules. I t allows for hydrogen bonds between the two CMP residues in positions 74 and 75 (see ref. 22) and the two GMP residues in positions 20 and 21. The t R N A structure is further stabilized b y a hydrogen bond between the AMP residue in position 73 and the T 54 in the T}FGC loop. From the results presented here it appears t h a t in the deacylated state of t R N A the two CMP residues of the -pCpCpA stem interact strongly with some other region of the t R N A molecule. Our data thus support a conformation of t R N A as proposed b y CRAMER et al. ~9 After aminoacylation of t R N A the rate of the venom phosphodiesterase-mediated release of the terminal AMP decreased b y a factor of 5- The product of this reaction is a 3' (2')-0-aminoacyl-AMP (to be published). This is consistent with an exonucleolytic attack on aminoacyl-tRNA. YOT et al. s also observed that ammoacyl-tRNA is a substrate for venom phosphodiesterase. However, these authors did not observe a decrease in the rate of hydrolysis after aminoacylatlon of tRNA. Also, they did not observe any differences between particular aminoacyl-tRNA species while we saw significant variations between five aminoacyl-tRNA's tested. YOT et al. s claimed there was no difference between the rate of venom phosphodiesterase-medlated hydrolysis of deacylated t R N A and N-acetylvalyl-tRNA. The differences between the present work and that of YOT et al. s might well be due to the more indirect experimental approach of the latter authors. Furthermore, the lability at alkaline p H of the ester bond in aminoacyl-tRNA varies for particular amino acids. RAZZELL AND K H O R A N A 3°, showed that venom phosphodiesterase is sensitive to the environment around the 2'- and 3'-OH group of the rlbosyl residue. The different rates of the venom phosphodiesterase-mediated release of aminoacyl-AMP reported here would then be expected since various amino acids confer different sterlc and inductive effects at the 2' or 3' position of the ribosyl moiety. It thus can be assumed that esterlflcation of the 2 ' ( 3 ' ) - 0 H of the terminal adenosyl residoe is the main reason for the decrease in rate of hydrolysis b y venom phosphodlesterase. Whether a conformatlonal change of t R N A after ammoacylatlon also contributes to this decrease cannot be decided from these experiments.
ACKNOWLEDGMENTS
This work was supported by research grants from the National Institutes of Health (GM I3364), U.S. Public Health Service; and the American Cancer Society ( P - 5 I I ) . J . P . M . is the recipient of a Predoctoral fellowship from the National Institutes of Health (GM 42897), U.S. Public Health Service. M. E. H.-B. is the recipient of a Traineeship from the National Institutes of Health (GM 446), U.S. Public Health Service. The technical assistance of Mrs. C. Lemp is gratefully acknowledged.
REFERENCES i 2 3 4
J J. W T
PREISS, 1V~ DIECKMANN AND P. BERG, J B*ol Chem, 236 (1961) 1748. J FURTH, J HURWlTZ, R. KRUG AND M ALEXANDER, ] Bzol Chem., 236 (1961) 3317. E RAZZELL AND H C*. KHORANA, J B w l Chem, 234 (1959)2114 NIHEI AND G L CANTONI, ] Bwl. Chem , 238 (1963) 3991. Bzoch*m B*ophys. Acta, 217 (197 o) 176-188
J . P . MILLER et al.
188
ZUBAY AND ~I. TAKANAMI, B*ochem Bzophys. Res, Commun., 15 (I964) 207 ROSSET, R. MONET AND J JULIEN, Bull Soc Chzm. B~ol, 46 (1964) 87 . B KELLER, Bzochem. Bzophys. Res. Commun, 17 (1964) 412 YOT, P GUEGUEN AND F. CHAPEVILLE, F E B S Letters, i (1968) 156 LOWRY, N t~OSEBOROUGH, A FARR AND R RANDALL, J. Bzol Chem, 193 (1951) 265. VON EHRENSTEIN AND G LIPMANN, Proc Nail Acad. Scz U S , 47 (1961) 941 P. MILLER AND G R PHILIPPS, Bzochem B*ophys Res Commun, 38 (197 ° ) 1174 H ~¢~UENCHAND P ]~]~RG, in G L CANTONI AND D. R DAVIES, Procedures zn Nucleic Aczd Research, H a r p e r and Row, New York, 1966, p. 375 G R PHILIPPS, J Bzol Chem., 245 (197 ° ) 859 J L. KOERNER AND R L SINSHEIMER, J B*ol Chem , 228 (1957) lO89 N SUEOKA AND J. HARDY, Arch. Bzochem B*ophys, 125 (1968) 558 U. LAOERKVlST AND P. BERG, J Mol. Bzol, 5 (1962) 323 IR MONIER AND M. CrRUNBERG-MANAGO, in Ac,des R,bonucle~ques el Polyphosphates, Colloq. Intern, Centre Natl Rech Se* Par*s, (1962) 163 J N. VOURNAKIS AND H. A. SCHERAGA, B*ochem~stry, 5 (1966) 2997. G G BROWNLEE AND F. GANGER, J. Mol B , o l , 23 (1967) 337 V DANIEL AND U. Z LITTAUER, J Mol B~ol, i i (1965) 692 J R FRESCO, R. D BLAKE AND R. LANGRIDGE, Nature, 220 (1968) 1285 G R PHIL1PPS, Nature, 223 (1969) 374J A LAKE AND W. W. BEEMAN, J. Mol Bzol, 31 (1968) 115 C R. CANTOR, S R JASKUNAS AND I TINOCO, J Mol B,ol, 20 (1966) 39 lc~. P DOCTOR, W. FULLER AND ~NT.L. \VEBB, Nature, 221 (1969) 58 J. NINO, A FAVRE AND 3/[ YANIF, Nature, 223 (1969) 1333 M LEVITT, Nature, 224 (1969) 759 G, MELCHER, F E B S Letters, 3 (1969) 185 F CRAMER, H DOEPNER, H VAN DER HAAR, E SCHLIMME AND H SEIDL, PFoc Natl .4cad $6,. U S., 61 (1968) 1384 \ ¥ E RAZZEL UND H G KHORANA, J Bzol Chem , 234 (1959) 21o5
5 G. 6 R. 7 E. 8 P 90 IO G. I I J. 12 t£ 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 :28 29 3°
B,och,m. Bzophys. Acta, 217 (I97 o) 176-188
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96590
ACTION OF VENOM P H O S P H O D I E S T E R A S E ON T R A N S F E R RNA FROM E S C H E R I C H I A
j
COLI
p. M I L L E R , M. E. H I R S T - B R U N S AND G. R
PHILIPPS
Department o[ B*ochemzstry, St L o i s Unzverszly School of Medicine, St. Lou*s, Mo. 631o 4 (U.S.A ) (Received March 23rd , 197 o)
SUMMARY
Digestion of t R N A by venom phosphodlesterase was assayed under a variety of conditions. Released nucleotldes were characterized and the amount determined b y chromatography on Dowex I-X8 The release was also determined b y readdltlon of AMP and CMP to digested t R N A usmg purified tRNA-nucleotmdyltransferase. The two methods gave identical results as long as the fourth nucleotlde from the 3'-OH terminus was not removed. The rate of removal of each of the three terminal nucleotides was determmed. The terminal AMP was removed about 4 times faster than the first CMP and 65 times faster than the second CMP. At 37 ° the rate was 3-5 tnnes faster than at 20 ° for all three nucleotides. No sigmficant differences were seen for a n y particular ammo acid-specific tRNA. The presence of an amlnoacyl group at the 3'(2') terminus of t R N A decreased the rate of hydrolysis of the terminal AMP by a factor of 5. Differences of the release of aminoacyl-O-AMP were seen for five different a m i n o a c y l - t R N A ' s tested.
INTRODUCTION
t R N A can serve as substrate for tRNA-nucleotidyltransferase (EC 2.7.7.25) after partial or complete removal of the 3'-terminal trinucleotide. This can be accomplished by limited digestion with venom phosphodiesterase (orthophosphorlc diester phosphohydrolase, EC 2.1. 4 I) from Crotalus adamanteus 1,2. The enzyme attacks t R N A from the 3'-OH end and consecutively hberates 5'-mononucleotides~,4. ZUBAY AND TAKANAMI5 a n d ROSSET et al 6 reported t h a t at 20 °, venom phosphodiesterase liberates only about I mole of AMP and 1.2 moles of CMP per mole of tRNA. At 37 ° degradation proceeds further. Under suitable conditions, the complete 3'-OH terminal trinucleotlde sequence can be removed a n d degradation continues beyond this point at a reduced rate 1,8,4,7. Here we report the results of a systematic study of the controlled digestion of Escherichm coh t R N A by venom phosphodmsterase. The rate of venom phosphodiesterase-medlated removal of each of the three terminal nucleotldes was determined at 20 and 37 °. We therefore conclude t h a t no conformatlonal change near the 3'-OH end of t R N A occurs between 20 and 37 °. We also compared the rate of hydrolysis of the terminal adenosyl residue of Bzochz~n Bzophys Acta, 217 (197 ° ) 176-188
ACTION OF VENOM PHOSPHODIESTERASEON tRNA
177
aminoacylated and deacylated tRNA. It had been reported s that the presence of an aminoacyl group in the 3'(2') position of the terminal adenosine of t R N A does not affect the rate of hydrolysis of tRNA by venom phosphodlesterase. Under the more stringent conditions used here differences in rate of the removal were observed.
MATERIALSAND METHODS 3H-labeled triphosphate nucleosides and E14C]amlno acids were obtained from Schwarz BioResearch, Inc., Orangeburg, N. Y. ATP, CTP, and Dowex I-X8 were from Calblochem, Los Angeles, Calif. Bis-p-nitrophenyl phosphate was from Sigma Chemical Company, St. Louis, Mo. Glass fiber filters 934 AH were from Reeve Angel, Clifton, N. J. Protein was determined by the method of LOWRY et al. 9 using bovine selum albumin as standard. For the estimation of t R N A concentration, an average molecular weight of 26 ooo and an absorbance in 0.2 M NaCl of A1% "-,,60 am : 220 was used 1°.
Enzymes and R N A Venom phosphodlesterase (Lot No. 8KA) was purchased from Worthington Biochemical Corporation, Freehold, N. J. It was dissolved in IO mM magnesium acetate, IO ~o glycerol to give a I.O mg/ml solution and was stored at --20 °. No loss of enzymic activity was observed over a period of 4 months, tRNA-nucleotidyltransferase was purified as previously described 11. Aminoacyl-tRNA synthetases were prepared by a modification of the method of MUENCH AND BERG12. Cells from E. col~ Strain A-I 9 were used and ribosomes and t R N A removed by liquid phase polymer fractionation ~l. The lower phase obtained after (NHa)2SO 4 precipitation was dialyzed against phosphate buffer and chromatographed on DEAE-cellulose 1~. This synthetase preparation was free of tRNA-nucleotidyltransferase, ribonuclease I, and tRNA. t R N A was purchased from Schwarz BioResearch and purified on Sephadex G-Ioo (ref. 13) Purification of tRNA val will be described elsewhere. Venom phosphodiesterase was assayed by a modification of the method of KOERNER AND SINSHEIMER 14. The increase with time of Aa0o nm was determined for a o.52-ml reaction mixture containing I I #moles bis-p-nitrophenyl phosphate, 21/,moles glycme-NaOH (pH 8 7), 6 #moles magnesium acetate, and lO'-2o #g venom phosphodiesterase. One enzyme unit was defined as that amount of protein which liberated one nmole p-mtrophenol per rain at 25 °.
Venom phosphodiesterase digestzon o / t R N A and chromatographic analys~s o/nucleot,des The Incubation mixture contained in 0.5 ml: 20 #moles glycine-NaOH (pH 8.7), 5 #moles magnesium acetate, 147 nmoles tRNA, and 5 o - 5 o o # g venom phosphodiesterase (147 unlts/mg). After incubation for different times, the mixture was apphed to a 0. 9 cm ×5o cm column of Sephadex G-25 with a layer of 0.2 g acidwashed sllicic acid 15 (IOO mesh) on top. Immediately before the sample was applied, 5 ml of IO mM Tris-HC1 (pH 7-3), I mM magnesium acetate, 300 mM NaC1 was run onto the column. The column was then eluted with o.I M sodmm acetate (pH 4-4) at 0.2 ml/min. After the t R N A peak had been eluted, the outlet of the Sephadex column was connected to the inlet of a I cm × 9 cm Dowex I-X8 (400 mesh) column
B*och*m B~ophys. Acta, 217 (197o) 176-188
178
j . P . MILLER et al.
equilibrated with the same buffer. The low-molecular-weight peak from the Sephadex column was thus quantitatively transferred to the Dowex column. The Dowex column was washed with IO ml of o i M sodium acetate (pH 4.4) and then eluted with a linear gradient of 5 ° m l o.I M sodium acetate (pH 4.4) and 50 ml 3.o M sodium acetate (pH 4.4) The A2¢0 nm of the eluate was monitored and recorded. The elution volume and the area under each peak were determined for each nucleotlde. They were identified by their A2s0 nm/A260 nm ratio. A mixture of known amounts of the major nucleotides and adenosine was chromotographed on an identical column. Recovery was greater than 95 %- The nucleotldes released after venom phosphodlesterase digestion were quantltated b y direct spectrophotometrlc analysis or by comparison of the area under each peak with that of the standard column The two methods gave essentially the same results. The t R N A from the Sephadex column was used in the assays described below
Enzymatic add#,on o/nucleotides to venom phosphodiesterase d,gested t R N A Extent of t R N A digestion was also determined by enzymatic readdltlon of nucleotldes. Fractions from the Sephadex column containing t R N A were pooled and precipitated with ethanol The t R N A was collected by centrlfugation and the pellet dissolved in I mM Tns-HC1 (pH 7-5), IO mM magnesium acetate. The assay mixture contained in o.I ml: 5/,moles glycine-NaOH (pH 9.2), I / , m o l e magnesium acetate, I/~mole GSH, 0.2-0.6 nmole venom phosphodlesterase-treated tRNA, 20 #g bovine serum albumin, and I 4-4.2 units (1-3/zg) tRNA-nucleotldyltransferase. For AMP incorporation the mixture also contained lO.4 nmoles (12.4 nC) of [8-3HIATP and 5 6 nmoles CTP; for CMP incorporation it contained 15. 4 nmoles (23.2 nC) of [5-aHICTP either in the presence or absence of 60.0 nmoles ATP. For the incorporation of GMP and UMP 19.6 nmoles (18. 7 nC) E3HIGTP or 16.1 nmoles (15.9 nC) ~5-3HIUTP were used. All assays were done in duphcate. Incubation was for I h at 37 ° The reaction was stopped b y the addition of 2 ml of ice-cold 20 mM EDTA, 0.2 mM ATP and 2 ml of IO % trichloroacetlc acid. The precipitate was collected on glass fiber filters. Tubes and filters were washed twice with 3.5 % trlchloroacetic acid and the filters once with 95 % ethanol. They were dried and counted in 3.0 ml of a toluenebased scintillator in a Packard 3380 liquid scintillation spectrometer. To produce larger amounts of labeled tRNA, 14o nmoles venom phosphodiesterase-treated t R N A and IOO units tRNA-nucleotidyltransferase were incubated in 2 ml of the same buffer. Either 2.48/,moles (2.95 pC) ~8-3HlATP and 0.63/~moles CTP or o.7o/~moles (1.o5/~C) [5-aHICTP and 2.80 /,moles ATP were used After Incubation for I h at 37 °, the mixture was applied to a Sephadex G-25 column equilibrated with IO mM Trls-HC1 (pH 7.3), I mM magnesium acetate, 0.3 M NaC1; with 0.5 g acid-washed silicic acid layered on top. Fractions containing t R N A were pooled precipitated with ethanol, and the precipitate dissolved in I ml of I mM Tris-HC1 (pH 7 5), IO mM magnesium acetate Determination o/amino acid acceptor act,wty A typical reaction mixture contained in 7 ° # 1 : 7 5 / , m o l e s sodium cacodylate (pH 7.2), 7.5/,moles of KC1, 1.2/~moles of magnesium acetate, o 175/~mole ATP, 0.725/zg bovine serum albumin, 16o pmoles of tRNA, 9/~g crude ammoacyl-tRNA synthetases, 80 pmoles of [14Clamlno acid, and 6.4 nmoles of the other nineteen Bzoch,m Bzophys. Acta, 217 (197o) 176-188
ACTION OF VENOM PHOSPHODIESTERASE ON t R N A
179
a m i n o acids. Th e r e a c t i o n m i x t u r e was i n c u b a t e d for IO m i n at 37 °, s t o p p e d b y ad d i tion of 0.5 ml of 7 ~o trichloroacetic acid c o n t a i n i n g an excess of u n l ab el ed a m i n o acids, a n d t h e p r e c i p i t a t e d t R N A collected on glass fiber filters. Th e filters were washed w i t h trlchloroacetic acid a n d ethanol, dried an d c o u n t e d in 3 ml of a toluenebased scintillator.
RESULTS
E]/ect o/ temperature and time on venom phosphodiesterase digest*on T h e e x t e n t of digestion of t R N A b y v e n o m phosphodiesterase was assayed b y analysis of the nucleotides released. N u c l e o ti d e s a n d t R N A were s e p a r a t e d on Sephade x G-25 an d t h e nucleotides s e p a r a t e d on D o w e x I - X 8 . Fig. I shows the results of a 2o ° digestion. Th e a m o u n t of A M P released from t R N A increased r a p i d l y a n d reached a m a x i m u m after I h. CMP was r e m o v e d at a n o t i c e a b l y slower rate. T h e increase in CMP r e m o v a l after 4 h p r o b a b l y r ep r esen t ed release of t h e t h i r d nucleotide f r o m t R N A . A small a m o u n t of U M P was also d et ect ed , while no G M P was released. No adenosine was f o u n d in t h e low-molecular-weight fraction f r o m the S e p h a d e x column. This suggested t h a t 5'-nucleotidase (EC 3.1.3.5) a n d rlbonuclease a c t i v i t y (EC 2.7.7.17) were absent. T h e t R N A recovered f r o m S e p h a d e x was st u d i ed for its ab i l i t y to i n c o r p o r a t e nucleotides e n z y m a t i c a l l y . The a m o u n t of A M P r e m o v e d b y v e n o m phosphodiester i s e e q u a l e d the a m o u n t t h a t could be a d d e d back. I n Fig. 2 t h e results of the enzy-
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Fig. I. Digestion of tRNA by venom phosphodmsterase at 20 ° Dlgestmn was performedIas described in MATERIALSAND METHODS using 14 7 unlts/ml venom phosphodiesterase and 294 nmoles/ml t:RNA. Nucleotldes released were assayed by chromatography on Dowex I-X8. × - ×, AMP removed, {} - 0 , CMP removed, O - Q , UMP removed Fig. 2. Addition of CMP by tRNA-nucleottdyltransferase to tRNA digested at 20 ° The tRNA was the same as used m F]g. i Nucleotlde addition was performed as described in MATERIALSAND METHODS using 2.8 units tRNA-nucleotldyltransferase and o 3 nmole tRNA per assay. { } - 0 , CMP loss as assayed by Dowex I-X8 chromatography (from Fig I), O- - -O, CMP addition an the presence of o.6o mM ATP; × - x , CMP addition m the absence of ATP.
B,och,m. Bwphys Acta, 217 (I97 o) i76-188
18o
j. P MILLER et al.
matic a d d i t i o n of CMP are shown. W h e n assayed in the absence of A T P a p p r o x i m a tely twice as m u c h CMP was a d d e d to t R N A as was released. This m a y be caused b y the f o r m a t i o n of t R N A - p C p C p C , a possibility which is c u r r e n t l y u n d e r investigation. W h e n a d d i t i o n was studied in the presence of 0.6 mM ATP, a d d i t i o n of CMP equaled the release. I n c o r p o r a t i o n of U M P was 0.2 mole/mole t R N A m all instances
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Fig. 3 Digestion of tRNA by venom phosphodlesterase at 37° The conditions were the same as m Fig I x - x , AMP removed, O - O , CMP removed, Q - O , GMP removed, O - - - 0 , UMP removed Fig 4 Addition of AMP by tRNA nucleotldyltransferase to tRNA digested at 37°. The tRNA used was the same as in Fig 3, 4 2 umts tRNA-nucleotldyltransferase and o 5 nmole tRNA were used per assay 0 - 0 , AMP loss as assayed by Dowex I-X8 (from Fig. 3), C) O, AMP addition. Fig. 3 shows the results of v e n o m phosphodlesterase digestion at 37 °. At the shortest time investigated, i e. after 30 rain, I mole of AMP a n d 1. 4 moles of CMP per mole of t R N A h a d been released. Thereafter, the release of CMP increased linearly up to a p o i n t where 2.2 moles of CMP h a d been removed. On the other h a n d , the a m o u n t of AMP In the nucleotide fraction r e m a i n e d c o n s t a n t for 4 h. Since AMP is f o u n d in the fourth position from the 3'-OH t e r m i n u s i n 69 ~o of E. coh t R N A a6, the increase after 4 h p r o b a b l y represents r e m o v a l of the fourth nucleotide. The time p o i n t where more t h a n I mole of AMP was released coincided with t h a t where 2 moles CMP were released. Both U M P a n d GMP appeared in the m o n o n u c l e o t l d e fraction from t R N A digested b y v e n o m phosphodiesterase at 37 °. I n Fig 4, the e n z y m a t i c r e a d d l t l o n of AMP was investigated. I mole of AMP was a d d e d to all t R N A digested for u p to 4 h. t R N A which h a d been digested longer accepted less AMP t h a n h a d been removed. Fig 5 shows a d d i t i o n of CMP to the same t R N A I n the absence of A T P a n extraneous a d d i t i o n of CMP was again seen, howBzochzm Bzophys Acta, 217 (197° ) I76-I88
ACTION OF VENOM PHOSPHODIESTERASE ON
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F i g 5 A d d i t i o n of C M P b y t R N A - n u c l e o t l d y l t r a n s f e r a s e t o t R N A d i g e s t e d a t 37 °. t R N A w a s t h e s a m e as t h a t used in Fig 3 O - Q , CMP loss as a s s a y e d b y c h r o m a t o g r a p h y on D o w e x I - X 8 (from Fig 3), C) - - - O , CMP a d d i t i o n in t h e p r e s e n c e of o 6o mM A T P , × - × , C MP a d d i t i o n in t h e ab sen ce of AT P.
ever the amount of CMP added above that which had been removed was only o.4 mole/mole tRNA. When CMP addition was assayed in the presence of ATP, incorporation equaled removal for up to 4 h. After that the abihty of t R N A to incorporate CMP decreased rapidly. Addition of UMP and GMP to tRNA digested at 37 ° was also investigated. No GMP could be incorporated regardless of the extent of digestion. Incorporation of UMP to tRNA gave values of approx. 0.25 mole/mole t R N A when digestion was up to 4 h; thereafter the values were slightly higher.
E//ect o/venom phosphodiesterase concentration on the extent o / t R N A hydrolysis The data of the preceding experiments suggested that the removal of the third nucleotide was very slow. To investigate this further, the removal of the third nucleotide was studied with higher venom phosphodiesterase concentrations. As shown in Expt. A of Table I, higher venom phosphodiesterase concentrations resulted in the removal of the third nucleotide, while the amount of AMP released remamed constant. All AMP and CMP residues which had been removed could be enzymatically reincorporated This shows that the complete -pCpCpA sequence can be removed at 20 ° but that larger amounts of enzyme and longer incubations are needed. At the highest enzyme concentration, incorporation of nucleotides into tRNA was less than the amount released. Comparing the AMP removal with the addition it is suggested that at least 5 % of all tRNA molecules had been digested beyond the third nucleotide. The addition of CMP lndmated that the third nucleotlde had been hberated from only about 80 % of all t R N A molecules While the amount of GMP released was proportional B,och*m. B~ophys. Acta, 217 (197 o) I 7 6 - I 8 8
J . p . MILLER et al.
182 TABLE
I
EFFECT OF VENOM PHOSPHODIESTERASE CONCENTRATION ON THE EXTENT OF DIGESTION OF t R N A t R N A w a s digested w i t h v e n o m p h o s p h o d l e s t e r a s e for 6 h a n d the nucleotldes r e m o v e d a n a l y z e d a s d e s c r i b e d u n d e r MATERIALS AND METHODS. T h e a s s a y f o r r e a d d l t l o n of A M P , G M P , C M P a n d U M P , u s i n g 2 8 u n i t s of t R N A n u c l e o t l d y l t r a n s f e r a s e a n d 0.2 n m o l e t R N A w a s p e r f o r m e d a s d e s c r i b e d in MATERIALS AND METHODS C M P i n c o r p o r a t i o n w a s d e t e r m i n e d in t h e p r e s e n c e of o 6 mM ATP.
Expt.
Temp
Enzyme conch (unzts/ml)
A
203
14. 7 44.1 73.5 148
B
37 °
147 44 I 735
Nucleotzdes removed (moles~mole t R N A )
Nucleot~des added (moles~mole t R N A )
AMP
CMP GMP
UMP AMP
C M P G3/IP U 3 I P
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I 08 I 96 271
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to the amount of enzyme, none could be reincorporated. Likewise, UMP was released in increasing amounts but remcorporation remained the same for all samples. In Expt. B of Table I, the results of a digestion at 37 ° are shown. At the lowest enzyme concentration all t R N A had lost the -pCpCpA terminus and IO % had lost four nucleotmdes. At an enzyme concentration of 44.1 umts/ml digestion had proceeded beyond the third nucleotide in 60-70 % as determined by CMP incorporation. The data suggested that approx, o. 7 mole of AMP was released from TABLE
I1
COMPARISON OF RATES ON VENOM PHOSPHODIESTERASI~ DIGESTION FOR EACH OF THE THREE TERMINAL NUCLEOTIDES OF t R N A
T h e t R N A - p C p C p [ S H ] A w a s p r e p a r e d b y a d d i t i o n of C M P a n d ESH]AMP t o i R M A - p C , t R N A pCp[3H]C was made by reconstituting iRMA-pC with [3H]CTP and ATP and redlgestmg the r e s u l t i n g t R N A t o r e m o v e m o s t of t h e t e r m i n a l A M P . T h i s t R N A c o n t a i n e d a p p r o x 45 % t R N A - p C p [ s H I C (15 % t R N A - p C p [ S H I C p A a n d 4 ° % i R M A - p C ) T h e t R N A - p E S H ] C w a s m a d e by reconstituting tRNA without CpCpA with [~H]CTP and ATP, the termmal dlnucleotlde w a s t h e n r e m o v e d . I n c u b a t i o n w a s a s d e s c r i b e d e x c e p t t h a t 2 o / 2 m o l e s of t R N A w e r e u s e d A h q u o t s of IO/21 w e r e r e m o v e d a n d p r e c i p i t a t e d w i t h t r l c h l o r o a c e t l e a c i d T h e a s s a y f o r b~s-pm t r o p h e n y l p h o s p h a t e w a s d e s c r i b e d i n MATERIALS AND METHODS
Substrate
Enzyme (t~g/ml)
Nucleotzderelease (pmoles/m*n p e r ~ g venom phosphod*esterase) 20 ° 37 °
Increase ~n rate at 37 ° over 2o ~ (-[old)
t R N A - p C p C p L3H7A
IO 3 2o 5 14 6 29 2 29.2 584
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Bzochzm. B~ophys. Acta, 217 (197 o) 1 7 6 - 1 8 8
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ACTION OF VENOM PHOSPHODIESTERASE
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183
the fourth position 16 while the remaining o 3 mole came from positions beyond the fourth nucleotide. To completely remove the fourth nucleotide, an enzyme concentration of 73-5 umts/ml had to be used. In this case, an average of 9 moles of nucleotides were removed. Therefore it might be assumed t h a t after the terminal trinucleotide was released t R N A was hydrolyzed at a faster rate. Release of UMP and GMP at 37 ° was also investigated. The amount increased with increasing enzyme concentrations When the fourth nucleotide was completely removed, 2.05 moles GMP and 1.59 moles UMP had been liberated. The amount of GMP and UMP released with 14. 7 umts/ml enzyme at 37 ° and 148 units/ml enzyme at 20 ° was the same Since under these conditions the amounts of AMP and CMP removed a p p r o x i m a t e l y equaled t h a t which could be added back, it appears t h a t GMP and UMP came from positions in t R N A t h a t do not effect the readition of the -pCpCpA terminus.
Rate o/removal o/term,nal nucleotides The results described so far suggested t h a t the three terminal nucleotides were removed from t R N A at different rates. In order to s t u d y these differences in greater detail, tRNA-pCpCpE3HIA, tRNA-pCpE3HIC, and tRNA-p[~HIC were prepared. The method for preparation is described in Table II. The rate of loss of 3H-labeled nucleotides was determined at 20 and 37 °. Two different enzyme concentrations were used and the rate was proportional to the enzyme concentration in each case. The terminal AMP was removed 3-4 times faster than the first CMP, and 50-80 times faster t h a n the second CMP. The increase in rate at 37 over 20 ° was 3-5-fold for all substrates. This showed t h a t the p r i m a r y difference between 20 and 37 ° is only a difference in rate and suggested t h a t the increase in velocity at 37 ° could be t o t a l l y accounted for b y an acceleration of the enzymatic catalysis. TABLE
III
AMINO ACID ACCEPTANCE OF RECONSTITUTED t R N A t R N A w a s d i g e s t e d 4 h a t 2 0 ° t o r e m o v e o n e A M P a n d t w o C M P r e s i d u e s ( C o l u m n 2) a n d d i g e s t e d 4 h a t 37 ° t o r e m o v e o n e A M P a n d t w o C M P r e s i d u e s B o t h t R N A ' s w e r e t h e n r e c o n s t i t u t e d . For amlnoacylatIon an enzyme preparation free of tRNA-nucleotldyltransferase was used.Inc u b a t i o n w a s f o r i o m m a t 37 °. T h e c o n t r o l t R N A w a s f r o m t h e s a m e l o t b u t u n t r e a t e d The asterisk indicates nucleotldes removed and added back
A ~nz~lo acid
Ala Arg Asp Glu Gly His Ile Leu Lys Met Phe Pro Set Tyr Val
A m , n o ac,d *ncorpor at*on (pmoles ) Control
tnXA-pCpC'pA"
tnNA@C'pC'pA"
47 35 12 18 7 19 16 87 23 37 25 12 25 22 55
49 26 16 II 4 19 17 46 17 36 27 15 14 15 42
5° 33 13 13 7 24 8 77 25 24 25 15 21 12 31
B*och*m. B*ophys. Acta, 2 1 7 (197 o) x 7 6 - - I 8 8
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184
Amino ac,d acceptance o/ reconstituted t R N A To d e t e r m i n e w h e t h e r v e n o m p h o s p h o d l e s t e r a s e a t t a c k s all a m i n o acid accepting t R N A species, t R N A was digested w i t h v e n o m p h o s p h o d i e s t e r a s e to remove increasing a m o u n t s of t h e t e r m i n a l d m u c l e o t i d e . F o r t e n a m i n o acids tested, we o b s e r v e d t h a t the loss of a m i n o acid a c c e p t a n c e increased w i t h t h e progress of v e n o m p h o s p h o d l e s t e r a s e digestion. There were no gross differences b e t w e e n different a m i n o acids A m i n o acid a c c e p t o r a c t i v i t y of d i g e s t e d t R N A after r e c o n s t l t u t l o n was also t e s t e d (Table I I I ) . The a c c e p t o r a c t i v i t y was d e t e r m i n e d for t R N A d i g e s t e d at 20 ° to r e m o v e one A M P a n d CMP a n d a t 37 ° to r e m o v e one A M P a n d two CMP residues. B o t h t R N A were t h e n r e c o n s t i t u t e d a n d t h e a m i n o acid a c c e p t a n c e c o m p a r e d w i t h t h a t of an u n t r e a t e d s a m p l e (Column I). No significant loss of acceptor a c t i v i t y was observed w i t h respect to a n y a m i n o acid. These d a t a suggest t h a t no i n d i v i d u a l species of t R N A was p r e f e r e n t i a l l y d i g e s t e d b e y o n d the t h i r d nucleotlde a n d t h a t t R N A - n u c l e o t i d y l t r a n s f e r a s e reacts w i t h all a m i n o acid-speclhc t R N A specl,.S
Venom phosphodiesterase dzgest,on o/ ammoacylated tRNA I n t h e course of these studies we a t t e m p t e d to p r o t e c t b y a m i n o a c y l a t i o n md l v i d u a l a m i n o acid-specific species from e n z y m a t i c a t t a c k b y v e n o m p h o s p h o d l esterase. Since a m i n o a c y l a t i o n d i d not p r o t e c t t R N A from e n z y m a t i c digestion, the effect of v e n o m p h o s p h o d l e s t e r a s e on a m i n o a c y l - t R N A was i n v e s t i g a t e d m more detail, t R N A was labeled w i t h aH in the 3 ' - A M P residue a n d one aliquot was t h e n a m i n o a c y l a t e d . Using the same c o n c e n t r a t i o n of b o t h s u b s t r a t e s the loss of trichloroacetlc acid-precip]table r a d i o a c t i v i t y as [ a l l ] A M P or a m i n o a c y l - I S H ] A M P was d e t e r m i n e d (Fig. 6A). To a v o i d s p o n t a n e o u s h y d r o l y s i s of the a m i n o acid ester b o n d i n c u b a t i o n was done at 23 ° a n d p H 7 2 The e n z y m e c o n c e n t r a t i o n was higher in this
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Fig. 6 A Effect of v e n o m p h o s p h o d l e s t e r a s e on t h e release of E3HIAMP from d e a c y l a t e d a n d a m m o a c y l a t e d t R N A The c o n c e n t r a t i o n s were p e r ml 8 6 nnl ol e s d e a c y l a t e d tRlXlA or 8 o n m o l e s a m m o a c y l a t e d t R N A , 143/,g v e n o m p h o s p h o d l e s t e r a s e m 5 n m o l e s Trls-HC1 (pFI 7 2), i o nmol e s Mg a c e t a t e A h q u o t s of a p p r o x , i o o p m o l e s t R N A were r e m o v e d a t i n t e r v a l s of I mln, s p o t t e d ont o M l l h p o r e h l t e r s , s o a k e d in IO % t r l c h l o r o a c e t l c acid, dri e d a n d c o u n t e d The specific a c t i v i t y of t h e 8H-labeled t R N A w a s a b o u t 31oo c o u n t s / r a m per nmole. Q - Q , a mmoa c yl -t R 1Y A , O - O , d e a c y l a t e d t R N A B. E f f e c t of v e n o m p h o s p h o d l e s t e r a s e on t h e release of [3H]CMP from d e a c y l a t e d a n d a m m o a c y l a t e d t R N A The a s s a y w a s p e r f o r m e d as d e s c r i b e d for A e x c e p t t h a t t he t R N A w a s l a b e l e d w i t h [3H]CTP 18 n m o l e s / m l of d e a c y l a t e d or 14 n m o l e s / m l a m l n o a c y l a t e d tRlXIA were i n c u b a t e d w i t h 1 4 3 / , g / m l of v e n o m p h o s p h o d l e s t e r a s e I n c u b a t i o n w a s c a rri e d o u t for a t o t a l of 4 ° m m a n d a h q u o t s were r e m o v e d e v e r y 5 m m The specific a c t l v l t y of t h e 3H-labeled t R N A w a s a b o u t i 6 o o c o u n t s / r a m p e r n m o l e (7)-(7), a m m o a c y l - t R N A ; O - Q , d e a c y l a t e d t R N A .
B*och*m B~ophys. Mcla, 217 (197 o) 176-188
A C T I O N OF V E N O M P H O S P H O D I E S T E R A S E
185
ON tRNA
experiment in order to observe AMP release over a shorter time period. The initial rate for deacylated t R N A was about 5 times the rate for amlnoacylated t R N A . In Fig. 6B removal of the adjacent CMP by venom phosphodmsterase was compared for deacylated and aminoacylated tRNA. The assay conditions were the same except that the incubation was for 4 ° rain, thus the slope only appears steeper in Fig. 6B. Again, the rate of digestion was 4 times faster for deacylated tRNA. At I rain, 3 1 % of the terminal AMP and about 8 % of the following CMP were removed from deacylated t R N A while these values were 6 and 2 %, respectively, for aminoacylated tRNA. Thus, the ratio of the initial rate for deacylated v s . aminoacylated tRNA was approximately the same in both cases. This indicated that the presence of the amino acid group retarded the removal of the 3'-terminal AMP residue only. TABLE
IV
THE EFFECT OF VENOM PHOSPHODIESTERASE ON AMINOACYL-tI{k'~A t R N A w a s a m l n o a c y l a t e d a n d p u r i f i e d a s d e s c r i b e d m MATERIALS AND METHODS 16 n m o l e s o f t h e v a r i o u s [ 1 4 C ] a m l n o a c y l - t R N A ' s w e r e i n c u b a t e d i n i m l w i t h i o / z m o l e s T r l s - H C 1 ( p H 7 4), i o p m o l e s m a g n e s i u m a c e t a t e , 4 ° / * m o l e s KC1 w i t h o r w i t h o u t 2 0 p g of v e n o m p h o s p h o d l e s t e r a s e a t 37 °. T h e r e a c t i o n w a s s t o p p e d b y a d d i t i o n of I m l o f i o % t r l c h l o r o a c e t l c a c i d a n d t h e p r e c i p i t a t e collected on Mllllpore filters The radmactivity determined as described
Venom Incubatzon Spee,#c radzoactw~ty phospho- T~me (counts/m~n per nmole t R N A ) d~esterase (re,n) Phe-tRNA Leu-tRN.4 Val-tRNA
--+ + +
o 2o o io 20
1°24 868 lOO2 608 349
5533 5o49 5489 4235 3171
H~s-tRNA
Am*noacyltRNA Ala-tRNA (algal protein hydrolysate )
574 541 569 415 331
1121 9oo lO42 794 732
57 ° 46o 57 ° 400 34 °
3754 2917 3159 2616 1337
ioo 94 99 72 57
ioo 80 93 71 65
ioo 81 ioo 7° 59
ioo 78 84 7° 36
Recovery of radzoactw~ O, (%) - -
- -
+ + +
o
20 o IO 2o
ioo 84 97 59 34
ioo 90 97 75 56
Digestion of deacylated tRNA by venom phosphodiesterase was about the same for all amino acid-specific species. To see whether the presence of a particular amino acid on t R N A affects venom phosphodiesterase digestion the experiment of Table IV was performed. Both spontaneous hydrolysis and the venom phosphodiesterasemediated release of labeled amino acids are shown. Release of the labeled amino acids from t R N A in the presence of venom phosphodiesterase differed for particular amino acids and these differences were unrelated to the variations observed for spontaneous hydrolysis.
DISCUSSION
The rates with which the 3'-terminal nucleotides of tRNA are enzymatically released by venom phosphodiesterase differ greatly. It is five times faster for the 3'B~och~m Bwphys. Acta, 2 1 7 (197 o) 1 7 6 - 1 8 8
186
j . p . MILLERet al.
terminal AMP than for the tollowing CMP residue and 8o times faster than for the next (second) CMP residue. This is not due to differences in the susceptlblhty to phosphodlesterase action among the four major nucleotlde bases 4. Due to the large difference m the rate of removal of the two CMP residues, only the terminal dlnucleotlde is liberated with low enzyme concentrations at 2o °. This confirms the results of others 1,~,5. At high concentrations of enzyme, the complete pCpCpA sequence call be removed at 20 °. Thus, our results exclude the hypothesis of ZUBAY AND TAKANAMI5 that the differences in the removal of the terminal nucleotldes between 20 and 37 ° are due to a weakening of the secondary structure of tRNA. This is substantiated by the observations that no gross changes exist in melting curves 17or optical rotatory dispersion curves is of t R N A in Mgz+ between 20 and 37 °. Therefore, the increase in rate of venom phosphodiesterase digestion between 20 and 37 ° is caused by a general effect of temperature on the reaction, rather than on the t R N A substrate. The t R N A used in these studies had been purified by chromatography on Sephadex G-Ioo (ref IOO). As we will describe elsewhere (G. R. PrlILIPPS, J. L. TIMKO AND T. W. MUNNS, in preparation), the t R N A was not contaminated by 5-S RNA, it gave a characteristic broad band after polyacrylamide gel electrophoresis. It was thus surprising to find a small amount of UMP in all digests at 20 °. ROSSET et al 6 made a similar observation but attributed this to a contamination with 5-S RNA: UMP is the 3'-termmal nucleotide of 5-S RNA of E. coh 19 Our results exclude this possibility. Also, UMP was incorporated mto tRNA while GMP was not. Incorporation of UMP into tRNA by rat liver tRNA-nucleotidyltransferase has been reported 20. FRESCO et al. 2~ suggested that the conformation of all tRNA species must be similar in order to fulfill the same functional requirements. That this might be indeed so, at least with respect to the -pCpCpA sequence, is suggested from our results which showed that no difference exists in rate or extent of digestion between different amino acid-specific tRNA's. Also, in prehminary experiments in which digestion of tRNA~~et and tRNAIval was studied, no gross differences were detected The reason for the different rates of removal of AMP and CMP may be the very structure of tRNA. All tRNA's of known sequence can be arranged in a cloverleaf conformation. Comparison of these structures 22 shows the fourth nucleotlde from the 3'-terminus is never hydrogen bonded. Thus the secondary structure cannot be the cause of the difference in rates of hydrolysis. The tertiary structure of tRNA, which is understood to be the folding of the different loops and the -pCpCpA stem into a more compact conformation, may be responsible for these differences. Various models have been proposed for the conformation of t R N A which differ with respect to the folding of the cloverleaf. LAKE AND BEEMAN 23 a n d CANTOR et al. 24 proposed an interaction between the anticodon loop and the -pCpCpA stem. DOCTORet al. 25, NINO et al. ~6, and LEVITT27 propose an interaction of the T~PGC loop and the amino acid stem. In the model of MELCHER 28, a hydrogen bond is formed between the 3'-terminal AMP residue of deacylated tRNA and the T 54 of the T ~ G C loop (in the nomenclature proposed by PHILIPPS 22) At the same time the AMP is positioned in close vicimty to the bases of the antlcodon The conformation proposed by CRAMERet al. zg, and its modification by M. STAEHELIN (personal communication) allows for an interaction of the -pCpCpA stem with the dlhydrouridine loop. This structure was originally proposed for yeast Bzoch~m Bzophys Acta, 217 (197° ) 176-188
ACTION OF VENOM PHOSPHODIESTERASE ON
tRNA
187
t R N A Phe but can be apphed to all t R N A molecules. I t allows for hydrogen bonds between the two CMP residues in positions 74 and 75 (see ref. 22) and the two GMP residues in positions 20 and 21. The t R N A structure is further stabilized b y a hydrogen bond between the AMP residue in position 73 and the T 54 in the T}FGC loop. From the results presented here it appears t h a t in the deacylated state of t R N A the two CMP residues of the -pCpCpA stem interact strongly with some other region of the t R N A molecule. Our data thus support a conformation of t R N A as proposed b y CRAMER et al. ~9 After aminoacylation of t R N A the rate of the venom phosphodiesterase-mediated release of the terminal AMP decreased b y a factor of 5- The product of this reaction is a 3' (2')-0-aminoacyl-AMP (to be published). This is consistent with an exonucleolytic attack on aminoacyl-tRNA. YOT et al. s also observed that ammoacyl-tRNA is a substrate for venom phosphodiesterase. However, these authors did not observe a decrease in the rate of hydrolysis after aminoacylatlon of tRNA. Also, they did not observe any differences between particular aminoacyl-tRNA species while we saw significant variations between five aminoacyl-tRNA's tested. YOT et al. s claimed there was no difference between the rate of venom phosphodiesterase-medlated hydrolysis of deacylated t R N A and N-acetylvalyl-tRNA. The differences between the present work and that of YOT et al. s might well be due to the more indirect experimental approach of the latter authors. Furthermore, the lability at alkaline p H of the ester bond in aminoacyl-tRNA varies for particular amino acids. RAZZELL AND K H O R A N A 3°, showed that venom phosphodiesterase is sensitive to the environment around the 2'- and 3'-OH group of the rlbosyl residue. The different rates of the venom phosphodiesterase-mediated release of aminoacyl-AMP reported here would then be expected since various amino acids confer different sterlc and inductive effects at the 2' or 3' position of the ribosyl moiety. It thus can be assumed that esterlflcation of the 2 ' ( 3 ' ) - 0 H of the terminal adenosyl residoe is the main reason for the decrease in rate of hydrolysis b y venom phosphodlesterase. Whether a conformatlonal change of t R N A after ammoacylatlon also contributes to this decrease cannot be decided from these experiments.
ACKNOWLEDGMENTS
This work was supported by research grants from the National Institutes of Health (GM I3364), U.S. Public Health Service; and the American Cancer Society ( P - 5 I I ) . J . P . M . is the recipient of a Predoctoral fellowship from the National Institutes of Health (GM 42897), U.S. Public Health Service. M. E. H.-B. is the recipient of a Traineeship from the National Institutes of Health (GM 446), U.S. Public Health Service. The technical assistance of Mrs. C. Lemp is gratefully acknowledged.
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