7
Eur. J. Biochem. 67,215-221 (1976) Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3'-Terminus Hans STERNBACH, Mathias SPRINZL, John B. HOBBS, and Friedrich CRAMER Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen (Received March 22/May 17, 1976) 2'-Deoxy-2'-amino-cytidylic acid can be incorporated into position 75 of tRNAPhr from yeast by tRNA nucleotidyltransferase yielding tRNAPh'-C-C(2'NH2). tRNAPh'-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid and of mercuriacetic acid to yield the derivatives tRNAPh'-C-C(2'NHCOCHzBr) and tRNAPh"-C-C(2'NHCOCH2Hg+OH-). Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNAPhe-C and tRNAPh'- C-C as well as ATP and CTP. A covalent, isolatable 1 : 1 complex between tRNAPhe-C-C(2'NHCOCHzBr) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxy- mercuri benzoate. Transfer RNA interacts with a variety of proteins during protein biosynthesis. Some insight into such interactions with ribosomal proteins, aminoacyl- tRNA synthetases or tRNA nucleotidyltransferase may be gained by covalently linking the tRNA to these proteins. Correct binding of the substrates carrying the reactive group in the active site of the enzymes is an essential prerequisite for such affinity labelling experiments. By this method some ribosomal proteins [1-4] as well as some aminoacyl-tRNA synthetases [5,6] have been labelled. In most of these experiments the a-amino group of the amino acid attached to the tRNA had a reactive group added on to it. Apparently tRNAs possessing reactive groups at their 3'-terminus are especially suited for labelling aminoacyl-tRNA synthetases and tRNA nucleotidyl- transferases. Recently we reported the modification of the 3'-terminus of tRNA by incorporation of modified nucleotides with tRNA nucleotidyltransferase [7 - 111. Here we report the introduction of 2'-deoxy- 2'-amino-cytidylic acid instead of CMP into position Ahhreviations. tRNAPh"-C74-C75-A76 = tRNAPhe = phenyl- alanine transfer RNA; tRNAph'-C-C and tRNAPh'-C = tRNAPh' lacking the terminal AMP and AMP + CMP, respectively; tRNA- Phe-C-C(2'NH2) = tRNAPh' with 2'-deoxy-2'-amino-cytidylic acid in position 75; tRNAPhe-C-C(2'NHCOCH2Br) = tRNAPh"-C-C- (2'NH2) substituted with the bromoacetyl residue. Enzymes. Pancreatic ribonuclease (EC 3.1.4.22) ; TI ribonuclease (EC 3.1.4.8); phenylalanyl-tRNA synthetase (EC 6.1.1.20); tRNA nucleotidyltransferase (EC 2.7.7.25). 75 of tRNAPhe,its formation of a derivative with bromoacetic acid and its interaction with tRNA nucleotidyltransferase. MATERIALS AND METHODS Chemicals 2'-Deoxy-2'-aminocytidine 5'-triphosphate was prepared by the method of Hobbs et al. [12]. ATP and CTP were obtained from Boehringer (Mannheim, Germany), p-hydroxymercuribenzoate from Schu- chardt (Miinchen, Germany), Aquasol from New England Nuclear (St. Albany, Boston, U.S.A.), Fluo- rescamine from Hoffman-LaRoche (Bade, Switzer- land), brom0[2-~~C]acetic acid (55 Ci/mol) from Ra- diochemical Centre (Amersham, England), p-hydroxy- mer~uri['~C]benzoate (10 Ci/mol) from CEA (Gif- sur-Yvette, France) and ['4C]ATP (47 Ci/mol) and ['4C]phenylalanine of Stanstar grade from Schwarz/ Mann (Orangeburg, N. Y., U.S.A.). All other chem- icals used were of the highest purity available from commercial sources. The N-hydroxysuccinimide ester of bromoacetic acid was prepared by the method of de Groot et al. [13]. The corresponding labelled compound was prepared as follows: 4.54 pmol br~mo['~C]acetic acid in 250 pmol ethyl acetate were treated with 5 pmol dicyclohexylcarbodiimide in 100 p1 ethyl acetate and 5 pmol N-hydroxysuccinimide in 250 p1 ethyl acetate. After 10 h at 20 "C the solvent was removed by evapo-

Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

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Page 1: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

Eur. J. Biochem. 67,215-221 (1976)

Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3'-Terminus Hans STERNBACH, Mathias SPRINZL, John B. HOBBS, and Friedrich CRAMER

Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen

(Received March 22/May 17, 1976)

2'-Deoxy-2'-amino-cytidylic acid can be incorporated into position 75 of tRNAPhr from yeast by tRNA nucleotidyltransferase yielding tRNAPh'-C-C(2'NH2). tRNAPh'-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid and of mercuriacetic acid to yield the derivatives tRNAPh'-C-C(2'NHCOCHzBr) and tRNAPh"-C-C(2'NHCOCH2Hg+OH-).

Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNAPhe-C and tRNAPh'- C-C as well as ATP and CTP.

A covalent, isolatable 1 : 1 complex between tRNAPhe-C-C(2'NHCOCHzBr) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxy- mercuri benzoate.

Transfer RNA interacts with a variety of proteins during protein biosynthesis. Some insight into such interactions with ribosomal proteins, aminoacyl- tRNA synthetases or tRNA nucleotidyltransferase may be gained by covalently linking the tRNA to these proteins. Correct binding of the substrates carrying the reactive group in the active site of the enzymes is an essential prerequisite for such affinity labelling experiments. By this method some ribosomal proteins [1-4] as well as some aminoacyl-tRNA synthetases [5,6] have been labelled. In most of these experiments the a-amino group of the amino acid attached to the tRNA had a reactive group added on to it.

Apparently tRNAs possessing reactive groups at their 3'-terminus are especially suited for labelling aminoacyl-tRNA synthetases and tRNA nucleotidyl- transferases. Recently we reported the modification of the 3'-terminus of tRNA by incorporation of modified nucleotides with tRNA nucleotidyltransferase [7 - 111. Here we report the introduction of 2'-deoxy- 2'-amino-cytidylic acid instead of CMP into position

Ahhreviations. tRNAPh"-C74-C75-A76 = tRNAPhe = phenyl- alanine transfer RNA; tRNAph'-C-C and tRNAPh'-C = tRNAPh' lacking the terminal AMP and AMP + CMP, respectively; tRNA- Phe-C-C(2'NH2) = tRNAPh' with 2'-deoxy-2'-amino-cytidylic acid in position 75; tRNAPhe-C-C(2'NHCOCH2Br) = tRNAPh"-C-C- (2'NH2) substituted with the bromoacetyl residue.

Enzymes. Pancreatic ribonuclease (EC 3.1.4.22) ; TI ribonuclease (EC 3.1.4.8); phenylalanyl-tRNA synthetase (EC 6.1.1.20); tRNA nucleotidyltransferase (EC 2.7.7.25).

75 of tRNAPhe, its formation of a derivative with bromoacetic acid and its interaction with tRNA nucleotidyltransferase.

MATERIALS AND METHODS

Chemicals

2'-Deoxy-2'-aminocytidine 5'-triphosphate was prepared by the method of Hobbs et al. [12]. ATP and CTP were obtained from Boehringer (Mannheim, Germany), p-hydroxymercuribenzoate from Schu- chardt (Miinchen, Germany), Aquasol from New England Nuclear (St. Albany, Boston, U.S.A.), Fluo- rescamine from Hoffman-LaRoche (Bade, Switzer- land), brom0[2-~~C]acetic acid (55 Ci/mol) from Ra- diochemical Centre (Amersham, England), p-hydroxy- mer~uri['~C]benzoate (10 Ci/mol) from CEA (Gif- sur-Yvette, France) and ['4C]ATP (47 Ci/mol) and ['4C]phenylalanine of Stanstar grade from Schwarz/ Mann (Orangeburg, N. Y., U.S.A.). All other chem- icals used were of the highest purity available from commercial sources.

The N-hydroxysuccinimide ester of bromoacetic acid was prepared by the method of de Groot et al. [13]. The corresponding labelled compound was prepared as follows: 4.54 pmol br~mo['~C]acetic acid in 250 pmol ethyl acetate were treated with 5 pmol dicyclohexylcarbodiimide in 100 p1 ethyl acetate and 5 pmol N-hydroxysuccinimide in 250 p1 ethyl acetate. After 10 h at 20 "C the solvent was removed by evapo-

Page 2: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

216 Affinity Labelling of tRNA Nucleotidyltransferase

ration and the residue was extracted three times with 20 p1 of ethyl acetate. The pooled extracts were added dropwise to 0.5 ml petroleum ether (40- 60 "C frac- tion), and the mixture left over night at 4°C. The resultant crystals were collected by centrifugation and dried in a desiccator. The dry product was used with- out further purification [14].

The niercuriacetic acid was prepared according to Tilander [15] and was converted to its N-hydroxy- succinimide ester by the method of Folsch [16].

tRNA

tRNAPhe from yeast was prepared from commercial bulk tRNA (Boehringer, Mannheim, Germany) ac- cording to Schneider et al. [17]. tRNAP'"-C was pre- pared from tRNAPhe-C-C by periodate oxidation, elimination of the terminal C75 residue, and removal of the residual phosphate group with bovine alkaline phosphatase [I 81 and purified by column chrornatog- raphy on Sephadex A-25 according to Solfert et al. ~191.

Enzymes

Pancreatic ribonuclease, 2 mg/ml, was a product of Boehringer (Mannheim, Germany) and TI ribo- nuclease of Sankyo Inc. (Tokyo, Japan). Phenyl- alanyl-tRNA synthetase from commercial baker's yeast (1 820 units/mg protein) was purified according to the procedure of von der Haar [20], tRNA nucleo- tidyltransferase from commercial baker's yeast was purified as described previously [21] ; the procedure was slightly modified and led to an enzyme with a specific activity of 44000 units/nig protein.

Incorporation qf 2'-Deoxy-2'-nnzinoc~tidylic Acid into tRNAPhe-C

The reaction mixture (2 ml) containing 100 mM KCl, 100mM Tris-HC1 pH9.0, 10mM MgS04, 0.5 mM mercaptoethanol, 0.3 mg/ml bovine serum albumin, 1.5 mM 2'-deoxy-2'-aminocytidine 5'-tri- phosphate, 200 A260 units tRNAPh"-C and 50 pg tRNA nucleotidyltransferase was kept at 32 "C for 2 h. Then 0.2 ml 2 M sodium acetate buffer pH 4.5 were added, the mixture diluted with 6 ml water and applied to a column of Sephadex A-25 (1 x 5 cm) equilibrated with 500 mM NaCl in 20 mM sodium acetate pH 5.2. The column was washed with 100 ml of the same buffer, then with 1.0 M NaCl in 20 mM sodium acetate pH 5.2. tRNA eluted at high ionic strength was precipitated with ethanol and collected by centrifuga- tion at 5000 rev./min for 20 min. The pellet was dissolved in 10 ml water and desalted on a Biogel P-2 column ( 3 x 60 cm) using water as solvent. Yield 180 A260 units tRNAPhe-C-C(2'NH2).

Acetylat ion of' tRNA Phe-C-C( 2'NHz) with Derivatives of [ 14C]Acetic Acid

To 60 A260 units of tRNAPh'-C-C(2'NH2) dissolved in 150 pl 200 mM triethanolamine-HC1 pH 8.0 was added the corresponding acetic acid derivative. In the case of br~mo['~C]acetic acid a 50-fold excess, in the case of unlabelled bromoacetic acid or hydroxy- mercuriacetic acid a 1000-fold excess, was used. After 2 h at 0 "C, 35 pl of dichloroacetic acid and 2.25 ml ethanol were added. The mixture was kept at -20 "C for 4 h and then centrifuged at 10000rev./min for 20min. The pellet of tRNA was washed with 2ml 75 :{ ethanol, then twice with 96 :< ethanol, and finally dissolved in 5 ml water and passed through a Biogel P2 column eluted with water. The tRNA fractions were concentrated by flash evaporation at 20 "C. The yield of the respective acetylated tRNAs was in all cases about 45 AZG0 units. The amount of unreacted tRNAPh'-C-C(2'NH2) in the reaction product was determined fluorimetrically using a fluorescarnine reagent [22]. The yields were 85 :{ of tRNAPhe-C-C- (2'NHCOCHzBr), 72 yd of tRNAPh'-C-C(2'NHCO- CHzHg'OH-) and 41 '4 of tRNAPh"-CC(2'NHCO- ['4C]CH~Br).

Analysis of the 3'-terminal nucleotides of tRNAPhe was performed on a nucleoside analyser [23]. 10 units of tRNA were incubated with 5 Fg pancreatic RNAse in 50 p1 200 mM ammonium acetate pH 7.0 for 2 h and then applied to the cation-exchange column of the analyser (Beckman M71). Oligonucleo- tides and nucleotides are eluted in the void volume, whereas the nucleosides originating from the 3'-end of tRNA are retarded and eluted later. The identifica- tion and quantitative determination of these nucleo- sides were performed as described [7].

Aminoacylation

The aminoacylation was performed in an incuba- tion mixture containing 0.15 M Tris-HC1 pH 7.65, 0.2 M KCI, 50 mM MgS04, 0.2 mM ATP, 0.2 iiiM CTP, 0.02 mM ['4C]phenylalanine, 3.5 mM bovine serum albumin and 70 mM niercaptoethanol. 0.2 A260 unit of tRNA in 100 pl incubation mixture was preincubated at 37 "C with 2 pg of tRNA nucleotidyl- transferase and the reaction was started with 0.5 unit of phenylalanyl-tRNA synthetase. 10-yl samples were removed after appropriate times up to 20 min and the tRNA-bound radioactivity was determined.

Assay for AMP Incorporation into tRNAPhe-C-C

The reaction mixture, unless indicated otherwise, contained in a final volume of 0.1 ml 10 pmol Tris- HC1 pH 9.0, 10 pmol KC1, 1 pniol MgS04, 2.0 A Z ~ O units of tRNAPhe-C-C, 50 nmol [14C]ATP (about

Page 3: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

H. Sternbach, M . Sprinzl, J. B. Hobbs, and F. Cramer 217

12000 counts min-' nmol-') and varying amounts of tRNA nucleotidyltransferase. Incubation was carried out at 32 "C for different times, after which normally 10 yl of the mixture was spotted on a Whatman 3MM paper disc. The discs were washed twice during 10 min with 5 yd trichloroacetic acid and finally with ethanol and ether, respectively. After drying the tRNA- bound radioactivity was determined in a Packard Tricarb liquid scintillation counter model 3375.

For all inhibition experiments tRNA nucleotidyl- transferase was incubated with the given amount of the respective inhibitor in the presence of 0.1 M Tris- HCl pH 9.0, 0.1 M KCl and 0.01 M MgSO4. Aliquots of the mixture, in most cases 1 pl, were transferred to I00 yl of the reaction mixture and the activity of the enzyme was determined as described above.

RESULTS

Preparation and Analysis ofPhe-tRNAP"-C-C(2"H2J-A

2'-Deoxy-2'-aminocytidine 5'-triphosphate is a substrate for tRNA nucleotidyltransferase from baker's yeast. Almost complete incorporation of this modified nucleotide into position 75 of tRNAPhe-C from yeast was achieved. tRNAP"e-C-C(2'NH2) could be further enzymically converted to tRNAPh'-C-C- (2'NH2)-A which is a good substrate for phenyl- alanyl-tRNA synthetase. The aminoacylation of tRNAPhe-C-C(2'-NH2) performed in the presence of ATP and tRNA nucleotidyltransferase provides a very sensitive assay for the determination of the extent of modified nucleotide incorporated into the 3'- terminus of this tRNA (Table 1). Formation of

Scheme 1

Scheme 2

tRNAphC-C-C(2'NH2) and tRNAPhe-C-C(2'NHz)-A could also be demonstrated by 3'-end group analysis of the reaction products [7].

Reaction of tRNA"h'-C-C(2'NH2) Lvith Acetic Acid Derivatives and 2-Mercaptctrthanol

tRNAPhe-C-C(2'NH2) was acetylated either by br~mo[ '~C]acet ic acid, unlabelled bromoacetic acid or p-hydroxymercuriacetic acid using the procedure of de Groot rt al. [I31 (Scheme 1). With a large excess of acylating agent (1 000-fold) 85 and 72 ?g acylation could be achieved with bromoacetic acid orp-hydroxy- mercuriacetic acid, respectively. In the case of the labelled compound the smaller excess of reagent used is probably the reason for only 41 y~;, acetylation.

2'-Deoxy-2'-bromoacetamidocytidine reacts rap- idly with sulfliydryl reagents (Scheme 2, Fig. 1 A, B). After treatment of tRNA ph'-C-C(2'NHC0['4C]CH2- Br) with 2-mercaptoethanol rapid reaction occurs on the 3'-terminal ribose; the nucleoside formed is again eluted at the same position as the mercaptoethanol- treated 2'-deoxy-2'-bromoacetamidocytidine (Fig. 1 B, D).

tRNA [I4C]Phe accepted

without CTP with CTP

pmol:Azho unit (":, control)

tRNAPhe'-C-C-A (control) 1500 (100) 1500 (100) tRNAPhe-C 20 (1.3) 1450 (96)

1380 (92) t RNAPh"-C-C(2'N H7) 1120 (75 )

t R N A w C y t 7 ' + 0 . t R b J A w c ~ t ~ ~

N-0-CO-R

OH NH2 0 OH YH

CO I R

OH NH I co I CH2-Br + HS-CH~-CH~-OH

co I CH~-SS-C~$- CHi-OH

Page 4: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

218 Affinity Labelling of tRNA Nucleotidyltransferase

0 20 40 60 80 100 120 T;me (rnin)

Fig. 1. Ana1ysi.y of the 3'-terminal nucleoside qf tRNA'"' species [7J . (A) 0.5A260 unit of 3'-deoxy-2'-bromoacetamidocytidine as a stand- ard (elution volume: 31.5 ml). (B) 0.5 A m unit of 2'-deoxy-2'- bromoacetamidocytidine after incubation (30 min. 20 " C ) with 100 mM mercaptoethanol (total volume of 50 pl). The thioether formed eluted after 71 min (21.5 ml). (C) 0.3 AzbO unit tRNAPh'- C-C(2'NHCO[L4C]CH~Br) in 100 pI 50 mM ammonium acetate pH 7.0 after incubation with 5 pg pancreatic RNase for 2 h. (D) 0.3 Azb0 unit of tRNAPhe-C-C(2'NHCO[14C]CHzBr) in 100 pl 50 mM ammonium acetate pH 7.0 after incubation for 2 h with 5 pg pancreatic RNase in presence of 100 mM mercaptoethanol. 0.5-ml fractions were collected, diluted with 6 ml Aquasol and the radioactivity was determined in a Packard scintillation counter model 3375 (X-X). Absorbance was measured at 260nm (- ) and 280 nm (~ ---)

Inactivation o j t R N A Nucl~otidyltvansferase

Our preliminary experiments have shown that tRNA nucleotidyltransferase from baker's yeast is very sensitive against reagents blocking sulfhydryl groups, such as N-ethylmaleimide, 2,2'-dinitro-5,5'- dithiodibenzoate and p-hydroxymercuribenzoate (Fig. 2). If the enzyme possesses -SH groups essential for its activity which are at or near the active site i t should be inactivated also by tRNAPhe-C-C(2'NH- COCHZBr) and tRNAPhe-C-C(2'NHCOCH2Hg+- OH-). During incubation at 32 "C no more than a 2-fold excess of these tRNA derivatives inactivated the enzyme with a half-life of a few minutes (Fig.2), whereas in the presence of alkylating agents iodoacet- amide and bromoacetic acid, whose chemical reactivity

- 60 -

x I ._ .- > ' 4 0 - +-

E, c

cn .- c $j 20 E n

B.

'.. .*- I

I 0 10 20 30

Time (min)

Fig. 2. Inactivation of tRNA nucleotidyltransferase. 0.1 nmol of enzyme was incubated with 0.19 nmol of tRNAPhe-C-C(2'NHCO- CHzBr) (A- A), 0.19 nmol of tRNAPh'-C-C(2'NHCOCH2Hg+) (e--- e) or 0.3 nmol of p-hydroxymercuribenzoate (.-a) in 100 p1 0.1 M Tris . HCI buffer pH 9.0 containing 0.1 M KCI and 0.01 M MgS04. At the given times the remaining activity of the enzyme was tested as described under Methods. For comparison the same amount of tRNA nucleotidyltransferase was incubated without any admixture ( x - x ) and in the presence of 0.3 nmol of iodoacetainide (-~- 0) and 0.3 nmol of bromoacetic acid (0 -o), respectively

I loot-.-.-.---.-.-.----*- \&

1 0 10 20 30

Time (min)

Fig. 3. Inactivation of tRNA nucleotidyltransferase in the presence of its substrates. 0.1 nmol of enzyme was incubated with 0.19 nmol of tRNAPhe-C-C(2'NHCOCH2Br) without any substrate ( Y. -~ x ) and in presence of 0.9 nmol of tRNAPh'-C (+~e) and 500 nmol of ATP (.A) and 500 nmol of CTP (A-A), respectively. 100 p1 of the incubation mixture contained 0.1 M Tris-HC1 pH 9.0, 0.1 M KCI and 0.01 M MgS04. At the times indicated the re- maining activity of the enzyme was tested as described

is similar to or even higher than the reactivity of bromoacetylated tRNA, no inhibition of the enzyme was observed under identical conditions.

The rate of inactivation decreased significantly in the presence of the natural substrates (Fig.3). A

Page 5: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

H. Sternbach, M. Sprinzl,

0.3

, ,' ,,

0.4 -0.3 -0.2 -0.1

B. Hobbs, and F. Cramer

0.1 0.2 0.3 0.4 0.5 0.6 li[Sl (W')

Fig.4. Inhibitorji effect oJ'tRNA'"'-C~-C'(Z'NHCO HzBr) in presence of various conc'entrations o j tRNAPh'-C on the C M P incorporation (Lineweaver-Burk plot) . 0.2 nmol of enzyme were incubated with 0.4 nmol of bromoacetylated tRNA in the presence of the given amounts of tRNAPh'-C. Incubation mixture (100 PI) contained 0.1 M Tris-HCI pH 9.0: 0.1 M KCI and 0.01 M MgS04

small excess of tRNAPhe-C or tRNAPhe-C-C protected the enzyme completely against the attack of tRNAPhe- C-C(2'NHCOCH2Br). Preincubation of tRNA nu- cleotidyltransferase (2.4 pM) with constant amounts of bromoacetylated tRNA (5.0 pM) in the presence of increasing concentrations of tRNAPhe-C resulted in an increase in the remaining activity of the enzyme (Fig. 4). As sholild be expected in a case of a competi- tion for the same binding site, the double-reciprocal plot of the remaining enzyme activity veysus competitor concentration gives a straight line. The apparent inhibitor constant which reflects the inhibitory effect of the natural substrate on the measured chemical reaction is near the K,,, of tRNAPhe-C-C for the enzyme. This indicates that both tRNA species compete for the same site which is most probably identical with the site to which the tRNA is bound during normal function of the enzyme.

ATP and CTP were also able to protect the enzyme against the attack of tRNAPhe-C-C(2'NHCOCHzBr). In a concentration near to the K, values of these triphosphates they reduced the inactivation to about 50:4 (Fig.3). It should be noted that protection by CTP was more effective than by ATP. All these results indicate a specific inactivation of tRNA nucleotidyl- transferase by tRNAPhe-C-C(2'NHCOCH2Br).

In order to quantify the interaction of tRNAPhe- C-C(2'NHCO['4C]CH2Br) with tRNA nucleotidyl- transferase the reaction product of tRNA (four parts) and enzyme (one part) was passed through a Sephadex G-100 column after incubation at 32 'C until the enzyme was completely inactivated. Gel filtration of the reaction mixture led to separation of a covalently linked complex between tRNA and tRNA nucleotidyltransferase from the excess of tRNA (Fig. 5). This complex, which eluted markedly earlier

- c ._ 5 10000 - c 2 0 " 1

.r" 6000 .- - 0 m

a 5 2000 w

500

c .~ E . m

300 :: - 0 -

100

1

L .- 5 300C rn

L 3 0 " 1

0 L?

1 00c

219

: 0.3 8

N +. a,

c m fl

0.1 - 9

Fraction number

Fig. 5. Sephader G-100 ,filtration .f t R N A ~~uc'1entid~1t~un.sf~ru.s~~ inhibitor complexes. (A) 0.74 nmol of enzyme and 1.6 nmol of tRNA""'-C-C after incubation at 32 "C for 30 min in 0.1 M Tria- HCI pH 9.0 containing 0.1 M KCI and 0.1 M MgS04 were passed through the column (1.5x60cm). In order to estimate where the iliitive enzyme and tRNAPh"-C-C elute, the enzyme activity was determined as described ( x - x ) and the absorbance at 260 nm was measured (O-- a). (B) 0.74 nmol of enzyme were incubated together with 1.95 nmol of tRNAPh'-C-C(2'NHCO- [I4C]CH2Br) under the conditions described in (A) until the enzyme was completely inactivated (60 min) and applied to the column. The elution of I4C was monitored ( x - ~~~ x ). (C) A mixture of 0.74 nmol of enzyme and 0.6 pmol of p-hydroxymercuri["C]- benzoate in the buffer described in (A), which was incubated at 32 "C until the enzyme was completely inactivated (60 rnin), was applied onto the column. The elution of I4C was monitored ( x ~~ ~ x ). The eluant in all cases was 0.05 M Tris-HCI pH 9 .0 containing 0.1 M KCI and 0.01 M MgS04. Fractions of 2.0 ml were collected

from the column, could be repeatedly passed through the column without loss of radioactivity. In order to calculate the stoichiometry of the reaction of tRNAPh'- C-C(2'NHCOCH2Br) with tRNA nucleotidyltrans- ferase the fractions of the peak which was eluted first were pooled and the amount of radioactivity was estimated. This experiment was repeated with different amounts of enzyme. The results in all cases indicate an incorporation of 1 - 1.3 mol of tRNA into 1 mol

Page 6: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

220 Affinity Labelling of tRNA Nucleotidyltransferase

Table 2. Formation of' contplexes of t R N A nucleo t id~l t ran .s f~~~~~se with tRNAPh'-C-C{2'NHCO( I4C]CHz Br) and p-hydro.uymercuri- [ ''C]benzoate, respectively

Enzyme tRNAPhe-C-C- p-Hydroxy- Ratio of (2'NHCO- mercuri- components [I4C]CH2Br) ['4C]benzoate bound bound

nmol

0.5 0.585 ~ 1 : 1.17 0.74 0.925 - 1 : 1.25 0.95 1.01 - 1 : 1.06 0.5 - 0.52 1 : 1.04 0.95 - 0.92 1 : 0.97

of tRNA nucleotidy~transferase (Table 2). Isolation of the product of reaction between the enzyme and p-hydro~ymercuri['~C]benzoate by gel filtration on Sephadex G-100 afforded a 1 : 1 complex in respect of the radioactivity bound to the enzyme.

In order to ascertain whether tRNAPhe-C-C(2'- NHCOCH2Br) and p-hydroxymercuribenzoate com- pete for the same sulfiydryl group, tRNA nucleotidyl- transferase was first inactivated with tRNAPhe-C-C- (2'NHCOCH2Br) and then incubated with p-hydroxy- mer~uri['~C]benzoate. In this case no radioactivity could be detected to be associated with the protein. The same result was obtained when the enzyme was incubated first with p-hydroxymercuribenzoate and then with tRNA'h'-C-C(2'NHCO['4C]CH2Br). Prob- ably there is only one reactive - SH group of a cysteine residue in or near the active site of the enzyme.

DISCUSSION

One of the proteins with which tRNA interacts is tRNA nucleotidyltransferase. This protein is capable of regenerating the -C-C-A end of tRNA using the triphosphates of cytidine and adenosine as substrates but has no specificity for particular tRNA molecules comparable with that of aminoacyl-tRNA synthetases. As part of a program to investigate proteins which interact with tRNA we intended to characterize the active centre of tRNA nucleotidyltransferase from yeast using derivatives of tRNA as well as analogues of ATP and CTP in affinity labelling experiments. A number of derivatives of aminoacyl-tRNA have been synthesized in the past in order to label either amino- acyl-tRNA synthetases [5,6] or ribosomal proteins of tRNA binding sites. They all suffer from the lability of the ester bond by which the amino acid bearing the reactive group is linked to the tRNA. With the avail- ability of 2'-deoxy-2'-aminocytidine 5'-triphosphate it seemed feasible to introduce 2'-deoxy-2'-aminocytidylic acid into position 75 of tRNAPhe and to react this modified tRNA with acetic acid derivatives. In this

way one would obtain a tRNA to which the reactive group is attached by a stable amide bond.

2'-Deoxy-2'-aminocytidine 5'-triphosphate is a substrate for tRNA nucleotidyltransferase and can be incorporated quantitatively into position 75 as well as position 74 (not shown) of tRNAPhe under con- ditions which are identical for the incorporation of CTP. In contrast, we had earlier tried unsuccessfully to incorporate 2'-chloro-2'-deoxy-cytidine 5'-triphos- phate in position 75 of tRNAPh'. Thus it is clear that there are limitations to the tolerance of the enzyme with respect to modifications in the 2'-position of CTP ; tRNAph"-C-C(2'NH2) is still a good substrate for tRNA nucleotidyltransferase which can incorpo- rate the terminal AMP thus leading to a modified but complete 3'-terminus. This tRNA can be amino- acylated quantitatively by phenylalanyl-tRNA syn- thetase. The results indicate that tRNA nucleotidyl- transferase interacts with tRNAPh"-C-C(2'NH2) and tRNAPhe-C-C in the same manner. This must be con- sidered as a prerequisite for affinity labelling experi- ments.

Acetylation of the 2'-amino group could be follow- ed by addition of fluorescamine [22]. Reaction of this reagent with primary amino groups results in an inten- sively fluorescing product. The measurement of this fluorescence represents a very sensitive method for the determination of the remaining free amino groups. Amino groups of adenosine, cytidine and guanosine do not react with fluorescamine (Sprinzl, unpublished results).

As shown by Carre et a/. [24], tRNA nucleotidyl- transferase from E. coli is sensitive to - SH-blocking reagents, whereas the enzyme from rat liver is not (M. P. Deutscher, personal communication). Since we find that tRNA nucleotidyltransferase from baker's yeast is also inactivated by - SH-blocking reagents its seems plausible that tRNAPh'-C-C-(2'NHCOCH2- Br) carries out inactivation by reacting preferentially with - SH groups although tRNAPh'-C-C(2'-NHCO- CHZBr) could in principle react with a number of amino acid residues of proteins such as glutamic acid [26], histidine [27], tyrosine, lysine [28] and methionine as well as cysteine [29]. The very fast inactivation of tRNA nucleotidyltransferase in the presence of concen- trations of tRNAph"-C-C(2'NHCOCH2Br) (2 pM), which are in the range of the K , value for tRNAP'"-C-C (5 pM), allows us to presume a specific reaction on the active site of the enzyme. The rate of inhibition is decreased by the simultaneous addition of tRNAPh' lacking its -C-C-A terminus partly or totally. This supports the latter assumption. The protection of the enzyme inactivation by its natural substrate tRNAs depends directly upon their concentrations. The en- zyme can also be protected by the two small substrate molecules, CTP and ATP. Their K , values are 0.2 mM and 0.6 mM, respectively. At these concentrations

Page 7: Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′-Terminus

H. Sternbach, M. Sprinzl, .I. B. Hobbs, and F. Cramer

they exhibit approximately 50 :< protection. These results indicate that the amino acid side chain reacting with the bromoacetyl group is in the vicinity of both the binding site for the 3'-terminus of tRNA and for the triphosphates.

p-Hydroxymercuribenzoate also inactivates the enzyme. Whether this inactivation is due to reaction of a - SH group at the active site or at a distant part of the protein cannot be decided. In the hope of increasing the specificity of this reaction we prepared tRNAPhe-C-C(2'NHCOCH2Hg+OH-). However, the kinetics of inhibition by the two mercury derivatives are similar. We cannot, therefore, draw any conclu- sions as to a reaction at the active site.

To investigate whether tRNAPhe-C-C(2'NHCO- CHIBr) reacted with an -SH group we first inactivated the enzyme with p-hydroxymercuribenzoate which also results in formation of a 1 : 1 covalent reaction product. The enzyme thus treated no longer reacted with tRNAP1"-C-C(2'NHCOCH2Br) as shown by the failure to isolate the corresponding product. The reverse experiment, first incubation with tRNAPhe- C-C(2'NHCOCH2Br) and subsequent reaction with p-hydroxymercuribenzoate, afforded the same result. The most logical explanation, we think, is that tRNAPhe-C-C(2'NHCOCH2Br) reacts with an - SH group of tRNA nucleotidyltransferase. The experi- ments with tRNAPhe-C-C(2'NHCOCHzBr) further indicate that this -SH group is at or near the active site of the enzyme. Whether this -SH group, which is essential for the enzyme activity, is directly involved in the nucleotide transfer reaction or only for binding of the substrates has to be clarified.

We are very grateful to W. Hanewacker for preparing tRNAP'Ie with complete or partially degraded C-C-A terminus. We wish to thank Dr F. Eckstein for many valuable discussions and Dr D . Gauss for critically reviewing the manuscript. The excellent technical assistance of Reinhild Engelhardt is gratefully acknowl- edged.

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H. Sternbach, M. Sprinzl, J. B. Hobbs, and F. Cramer, Abteilung Chemie, Max-Planck-Institut fur Experimentelle Medizin, Hermann-Rein-StraBe 3, D-3400 Gijttingen, Federal Republic of Germany