(C) 1991 by The American Society for Biochemistry and Molecular Biology, Inc THE JOURNAL of BIOLOGICAL CHEMISTRY Vol. 266, No. 34, Issue of December 5, pp. 22832-22836,1991

Printed in U.S.A.

Histidine tRNA Guanylyltransferase from Saccharomyces cereuisiae 11. CATALYTIC MECHANISM*

(Received for publication, July 1, 1991)

Dieter JahnS and Suchira Pande From the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 0651 1

Yeast histidine tRNA guanylyltransferase (TGT) catalyzes in the presence of ATP the addition of GTP to the 5‘ end of eukaryotic cytoplasmic tRNAHi” species. A study of the enzyme mechanism with purified protein showed that during the first step ATP is cleaved to AMP and PPi creating adenylylated TGT. In a second step the activated enzyme forms a stable complex with its cognate tRNA substrate. The 5”phosphate of the tRNA is adenylylated by nucleotide transfer from the adenylylated guanylyltransferase to form A(5’)pp(5’)N at the 5’-end of the tRNA. Finally, the 3“hydroxyl of GTP adds to th e activated 5‘ terminus of the tRNA with the release of AMP. This mechanism of tRNAHi” guanylyltransferase is very similar to that of RNA ligases. dATP can substitute for ATP in this reaction. Since among several guanosine compounds active in this reaction GTP is most efficiently added we believe that it is the natural substrate of TGT.

~ ~ ~ _ _ _ _ _

The extra nucleotide in position -1 of mitochondrial and eukaryotic cytoplasmic tRNAHis molecules is added posttran- scriptionally to the 5’ end of the tRNA by a histidine-tRNA- specific guanylyltransferase (Cooley et al., 1982; Williams et al., 1990; Abbe et al., 1990; Pande et al., 1991). Investigation of the catalytic properties of enzymes with similar functions demonstrated a variety of possible enzymatic mechanisms.

The mRNA guanylyltransferase, the mRNA-capping en- zyme, is another example of a posttranscriptional addition at the 5’ terminus of RNA. This enzyme adds a pG residue to the 5’ end of mRNA to form m7G(5’)pppN, the eukaryotic cap structure (Shatkin, 1976; Banerjee, 1980; Shuman and Moss, 1990; Shuman and Hurwitz, 1982). The multimeric yeast enzyme possesses three activities: a triphosphatase for the removal of the 5’-y-phosphoryl group of the mRNA, a guanylyltransferase for the guanylylation of the resulting 5’- diphosphoryl terminus, and a methyltransferase for its final methylation (Itoh et al., 1987). The enzyme is guanylylated (forming a enzyme-GMP complex) during the process.

A different catalytic mechanism is used by RNA ligase from T4-infected Escherichia coli cells (Uhlenbeck and Gumport, 1982). During the ligation reaction the 5”phosphate of the RNA molecule is adenylylated by the enzymes creating a 5‘- 5’-phosphoanhydride bond [A(5’)ppR(5’)], before the ligase forms the 3’-5’-phosphodiester linkage with the subsequent

* This work was supported by an NIH grant to Dieter Soll. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Dept. of Molecu- lar Biophysics and Biochemistry, Yale University, P. 0. Box 6666, New Haven, CT 06511. Tel.: 203-432-6204; Fax: 203-432-6202.

release of AMP. The enzymes get adenylylated during the process.

In order to understand the exact mechanism of the TGT’ and its relationship to the described catalytic processes we conducted a step-by-step analysis of the enzymatic function using purified protein. Here we describe the detailed mecha- nism of Saccharomyces cerevisiae TGT.

MATERIALS AND METHODS

GTP (400 Ci/mmol), [Y-~’P]ATP (3000 Ci/mmol), and [cI-~’P]ATP General-Biochemicals were reagent grade from Sigma. [CU-~’P]

(400 Ci/mmol) were purchased from Amersham. Unfractionated yeast tRNA was from Boehringer Mannheim. Sephadex G-50 and polynu- cleotides were purchased from Pharmacia LKB Biotechnology Inc. Precoated cellulose TLC plates were obtained from Merck. Polyeth- yleneimine (PE1)-cellulose TLC plates were from American-Tokyo Kasei Co.

Buffers-Buffers were as follows. Buffer A, 20 mM Hepes (pH 7.9), 10 mM MgCI,, 3 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, 50 mM NaCI, 10% glycerol; Buffer B, 20 mM Hepes (pH 7.9), 50 mM NaC1, 0.02 mM DTT, 1.5 mM MgC1’; Buffer C, 10 mM Tris (pH 8.0), 5 mM MgCl,, 0.3 M NaC1, 1 mM EDTA; Buffer D, 20 mM Hepes (pH 7.91, 10 mM MgC12, 3 mM DTT, 50 mM NaC1.

Assay for TGTActiuity-The employed exchange assay is described in the accompanying paper (Pande et al., 1991).

TLC Analysis of the Enzymatic ATP Cleauage-During pilot exper- iments we incubated the enzyme under standard conditions (buffer D) with radioactive ATP as described above. Besides AMP the reactions yielded another major product, perhaps the anhydride of AMP formed by the presence of alcohols in the assay mixture. To prevent the formation of this compound we dialyzed the enzyme against buffer B, excluding all alcohols and lowered the M$’ concen- tration in the assay. Moreover, the incubation time was shortened to 20 min. Finally dialyzed protein (5 pg of ATP-agarose fraction) was incubated in buffer B for 20 min a t 30 “C with the addition of 25 pmol of [32P]ATP, 200 pmol of nonradioactive ATP, and where indicated 1 mM GTP and 1 total yeast tRNA. After the incubation the reactions were mixed with nonradioactive AMP, ADP, and ATP as markers, dried down, redissolved in 20 p1 of water, and aliquots were spotted onto cellulose TLC plates. The plates were developed in isobutyric acid0.5 M ammonium hydroxide (5:3, v/v) (Silberklang et al., 1979). Labeled nucleotides were visualized by autoradiography

AMP, ADP, ATP (5 pg each) were visualized by UV light. and quantitated by scintillation counting. Nonradioactive markers of

Analysis of the Adenylylated TGT by Gel Filtration on Sephuder G50”TGT (2.5-5 pg of the Superose fraction) was incubated under standard conditions (buffer D) for 30 min at room temperature in the presence of either 1 mM nonradioactive ATP or 1 pCi of [cx-~’P]ATP or [Y-~’P]ATP. The radioactive ATP was diluted with nonradioactive ATP to a final concentration of 0.5 p ~ . Then the mixtures were loaded onto a Sephadex G-50 column (0.5 X 10 cm) equilibrated with buffer A and chromatographed with a flow rate of 10 ml/h. Fractions (200 ~ 1 ) were collected and subsequently quantitated by enzyme activity tests or scintillation counting. Vo was determined with Dex- tran blue. The position of the TGT and of ATP was analyzed in marker runs with purified enzyme (5 pg of Superose fraction) as

The abbreviations used are: TGT, tRNAH’” guanylyltransferase; DTT, dithiothreitol; HEPES, N-2-hydroxyethylpiperazine-W-2-eth- anesulfonic acid; TLC, thin layer chromatography.

22832

22833 Guanylyltransferase, Yeast, Histidine tRNA

standard and radioactive ATP as described ahove. IAhcling of I'lottrd Protrin on NitrocrUulose with Radioactive

ATP-Proteins ( I 5 pg of indicated fractions) were separated on a 10% SDS-polyacrylamide gel and semidry-hlotted onto nitrocellulose (Kyhse-Andersen, 1984). Nitrocellulose strips were incubated at room temperature for 1 h in 6 M guanidinium hydrochloride and suhse- ATP- m quently transfered into renaturation huffer (20 mM Hepes, pH 7.9, 10 mM MgCI,, 0.5% (w/v) hovine serum alhumin, 0.05% (w/v) Ficoll, 0.05% (w/v) polyvinyl pyrrolidone. 10 mM UTI', 10 mM CTP, 10 A2rd(1 r.

I,'. coli tRNA per ml). After five washes with renaturation buffer, Orlgln-

each for 30 min at 4 "C. 50 pCi of [tu-"T]ATP, 50 pCi of [ d ' P ] G T P F,c. 1. TGT ATP into AMP and PPI. purified TGT o r [-y-"'PJATI' were added and the incubation was continued over- (2 pg of protein of the superose fraction) wBs for 2o night. Finally the strips were intensively washed with renaturation at :jO oc under assay condition described under and buffer including 350 m M KCI, dried and the labelled proteins were ods.. using 25 pmol of [(,-,21)lATP (400 Ci,mmol) diluted with 200 visualized hy autoradiography (Schneider rt al.. 1989).

Isolation of tliNA"": 77;T Complrxrs hy Glycrrol (;radirnt Cmtrif- aliquots were spotted on TLC-cellulose plates were developed pmol of cold ATP and the additions listed helow. After the reaction.

ugntion-Analysis of protein-RNA complexes on glycerol gradients was performed as descrihed earlier (Jahn rt a/., 1990).

in isobutyric acid:0.5 M ammonium hydroxide = 5 3 (v/v). Detection

Ana/.vsis of the Adrnv/.v/atrd -5' End of tHNA""-Histidine tRNA dioactive ADP, and AMI, served as markers and were visualited of the :"I'-laheled AMP and ATP was hv autoradiography. Nonra-

was activated 1 ) ~ the incubation of T G T (2 PB of ATf'-agarose under uv light, Lane control reaction without enzyme conta in ing I'raction) with 0.02 AWW of purified yeast tRNA"'" for 1 h at room t R N A h and mM GTP; 2, complete reaction w i t h heat-inacti- temperature under standard conditions (huffer D) in the presence of vated TGT and the addition of mM GTI,; lanr *?, rr(;vr 1OpCi of [tu-:"I']ATI'. The labeled tRNA was isolated and quantitated alone without the addition of t R N A t ~ , e and GT1,; ,,, complete as descrihed ahove (Jahn et al., 1987). For the :,'-end characterization reaction with T(;T t h e addition of mM of the adenylylated tRNA"'", purified tRNA"" was laheled as de- scribed ahove and purified hy polyacrylamide gel electrophoresis. The RNAs were eluted from the gel hv soaking the excised gel slices for TARLE I 12 h at 42 "C in huffer C. The eluate was phenol extracted and the Nucleotide requiremmt for the TGT tliNAs collected hy ethanol precipitation and subsequently digested ~ 1 1 reactions using purified TGT (1-2 pg of ATP-agaroge fraction) hy RNase 1'1 and where indicated further treated with snake venom were carried out for 1 h at room temperature under standard rondi- phosphodiesterase. The liherated nucleotides were separated by pol- tions and quantitated as described under "Materials and Methods." yethyleneimine-cell~~lose TIL using 1 M ammonium formate (pH 3.5) we used the of the radioactive AMI' from the as solvent and visualized by autoradiography (Randerath et a[., 1980). 5' end of the tl{NA as measurement of the acceptance for different

fi;ffrct of Iliffrrrnl (;uanosinr Ilrriuatiurs in the G-addition Rrac- guanosine compounds hy TCT. ~ ' r p Was found most efficient, to t a l ly tion-Activation of the 5' end of the tRNA with radioactive ATP replacing the radioactive AMP on the and Was set was performed as described ahove. Different guanosine derivatives (1 wanylylation. The reductions of the of activated tRNA by mM) were added t.o the reaction and their effect was quantitated by other (1 m M ) were related to this value. the determination of the degreasing level of radioactivity incorporated into tRNA"" after separation on polyacrylamide gels, excision, and NTP (or tllllllo~) Cerenkov counting.

AMP- V ADP-

1 2 3 4

Other Ftnte of Rdditionn p p ~ n v l y l ~ t i n n

RESULTS

ATP Cleavage and Subsequent Adenylylation of the Enzyme Are the Frist Steps of the Guanylyltransferase Reaction-As described earlier for the Drosophila melanogaster and Saccha- romyces pombe enzymes (Cooley et al., 1982; Williams et al., 1990) and shown for the S . cereoisiae TGT in the accompa- nying paper (Pande et al., 1991) ATP is required for the enzymatic reaction. To investigate the role of ATP in the catalytic mechanism we analyzed by TLC the reaction of purified TGT (ATP agarose fraction) with [a-:"PP]ATP in the presence and absence of tRNA and GTP. We found tha t TGT alone cleaved ATP int.0 AMP and PP, (Fig. 1, lane 3 ) while ATP was stable under these conditions in the absence of enzyme (Fig. 1, lane I ) or in the presence of heat inactivated enzyme (Fig. 1, lane 2). The presence of tRNA and GTP did not stimu1at.e the ATP cleavage (Fig. 1, lane 4 ) . The enzyme is also able to use dATP but only 30% as well as ATP (see accompanying paper, Table 11). In contrast, the diphosphate ADP is not able t,o support the reaction. AMP and ADP inhibit the reaction almost completely (100 and 9496, respec- tively) when used in 7-fold excess (20 mM) over ATP. To address the question whether TGT gets adenylylated two different types of protein labeling experiments with [w:"P] ATP and [y-:"P]ATP were performed. Unincorporated nucle- otides and the enzyme were separated by gel filtration on Sephadex G-50. The positions of the free enzyme and of radioactive ATP were determined in control experiments by assaying enzyme activity or radioactivity in the column frac- tions (Fig. 2 A ) . First we incubated the native enzyme with radioactive ATP. The adenylylated form of the enzyme should be only detectable with [a-:"P]ATP while the phosphorylated

ATP 0 A T P G T P 1 0 ATP CDP ATP

55 G M P 13

ATP dGTP 85 ATP dCI)P 12

form is labeled with [y-"PIATP. Only in the presence of [ w "'PIATP radioactivity was detected in the position of the TGT, indicating the adenylylation of the enzyme and the release of the PPi after ATP cleavage (Fig. 2H). The release of PPi was analyzed independently by incubation of the enzyme with [y-"2P]ATP and subsequent analysis of the reaction products by TLC (data not shown). Moreover during the protein-labeling experiments no labeled TGT was de- tected with [y-""P]ATP (Fig. 2C). To ascertain that the TGT fraction was active we checked during both experiments for T G T activity without the addition of extra ATP. The enzyme activity was clearly detectable in both experiments without addition of ATP after gel filtration which removes a11 extra ATP, indicating a stable activated state of the enzyme induced by ATP cleavage (Fig. 2, R and C). A second series of protein- labeling experiments with the enzyme blotted onto nitrocel- lulose membrane linked the adenylylated enzymatic activity detected during the gel filtration experiments described above to the purified 58-kDa protein (see accompanying paper). For this purpose we used the high affinity of the enzymatic activity to ATP (discovered during ATP agarose chromatography, see accompanying paper). T G T in the 500 mM KC1 elution step from Heparin-agarose and a control fraction from the same column (350 mM KCI) devoid of TGT activity were blotted onto a nitrocellulose membrane after separation by SIh-

22834 Guanylyltransferase, Yeast, Histidinc tRNA

x I -

.I 5 I 9 I1 I 3 I5

Fracllon Number

FIG. 2. Activation of the guanylyltransferase by adenylyl- ation of the protein. A, ?KT (5 pg of protein of Superose fraction) clnd 25 pmol of [cr-'"I'jATP were chromatographed separately through Sephadex C;-50 gel filtration columns, 200-pl fractions were collected Hnd their elution position were determined by assaying enzyme activ- ity with addition of 3 mM A T P (0) and measuring radioactivity by scintillation counting ( X ) , respectively. R, T G T (2.5 pg of Superose f'raction) was incuhated with 1 pCi of [w:"P]ATP (400 Ci/mmol) diluted with cold A T P to a final concentration of 0.5 p~ and suhse- quently chromatographed through Sephadex G-50. Enzyme activity was determined under standard conditions as outlined under "Mate- rials and Methods" without the addition of A T P (0). The position of the radioactive ATP was determined by scintillation counting ( X ) .

ATP. (', same experiment as descrihed in C using [r-'"P]ATP for [n-:"P]

66-

4 3-

3 1-

FIG. 3. Labeling of the TGT polypeptide with radioactive ATP after renaturation on nitrocellulose membrane. Protein (15 p g ) of the 350 mM KC1 elution step (Innrs 1, 9. and 5 ) and the T G T activity containing 500 mM KC1 elution step (Innrs 2, 4. and 6 ) from heparin-agarose were separated on a 10% sodium dodecyl sul- fate-polyacrylamide gel and transfered to nitrocellulose. Blotted pro- teins were totally denatured by guanidine hydrochloride treatment and suhsequently renatured in the presence of [y-:"P]ATP ( h n r s 1 and 2 ) , [c~--"I']A'I'l' (Innrs 3 and 4 ) and [o-:"P]GTI' (Innrs 5 and 6). After intensively washing the blots, the laheled proteins were visual- ized hy autoradiography. T h e following marker proteins were used: myosin (200.000 I h ) , ($-galactosidase (116,000 Da), bovine serum alhumin (66,000 Da), ovalhumin (43,000 Da), and carhonic anhydrase (:11,000 Ua). The marker proteins were visualized hy Ponceau Red staining o f the nitrocellulose.

polyacrylamide gel electrophoresis. The blotted proteins were totally denatured by guanidine hydrochloride treatment and subsequently renatured in the presence of [n-:"P]ATP or [y- '"PIATP. Since T G T cleaves A T P t o A M P + PP, resulting in protein adenylylation, labeling of the correct polypeptide should only occur in the presence of [n-:"P]ATP. As shown in Fig. 3, lane 4, only in the activity containing fraction a

single protein of M, = 58,000 was labeled by the presence of

Activated Guanylyltransferase Stabl-y Rinds to tRNA""-To analyze the consequences of t he ATP cleavage for the further reaction sequence we investigated the stahilitv of t R N A . T C T complexes in dependence of ATP presence by glycerol gra- dient sedimentation. Gel-purified [n-.'T]G-laheled tRNA"" and purified TGT (ATP-agarose fraction) were incuhated under standard assay conditions without the addition of G T P and potential complexes were seaprated from the free nucleic acids by centrifugation through a 10455% glycerol gradient. The posit ions of free radioactive tRNA and free T G T were determined in parallel control gradients (Fig. 4, A and H ) . Only in the presence of A T P a shift of the labeled tRNA into the position of the enzyme caused by TGT. tRNA complex formation was detected (Fig. 4, compare C and I ) ) . Moreover, the result of the same experiment performed with labeled E . coli tRNA';'" without the detection of any complex formation served as control for the specificity of the hinding for yeast tRNA"'" (data not shown). This result demonstrates the ne- cessity of enzyme activation hy ATP cleavage prior to stable and specific tRNA"'" binding.

Activation of tRNA"" by Adenyl+ylation a t the -5' End, For- mation of Affj')ppfe5')N-Similar to our findings, adenylyla- tion of the enzyme and subsequent tRNA binding are part of the mechanism determined for RNA ligases investigated in the bacteriophage T4, in yeast and in wheat germ (Uhlenbeck and Gumport, 1982; Schartz et al., 1983; Xu et al., 1990; Pick et al., 1990). The reaction sequence for this kind of catalytic mechanism continues with the transfer of AMP to the 5' phosphate of the RNA molecule forming an activated A(5')pp5'(N) end. We analyzed the TGT reaction for the formation of an analogous intermediate by incuhation of the enzyme with tRNA and radioactive-labeled ATP in the ab- sence of G T P (Fig. SA, l a m I and 6). The analysis hy

[n-:"P]ATP.

I

10 20

Frsctlon Number JO

FIG. 4. Glycerol gradient analysis of the complex formed by the guanylyltransfcrase with tRNA"" in the presence of ATP. TG7' ( 2 0 pg of protein of ATI'-agnrose fraction) wns i n c r ~ h ~ ~ t e t l for 15 min at room temperature under standard assay conditions with the additions listed helow. The reactions were suhsequentlv centri- fuged through a 10-35% glycerol gradient for 22.5 h at 45,000 rpm in a Heckman SW50.1 rotor. After centrifugation, 15O-pl frartions were taken and analyzed for trichloroacetic acid-precipitable radionrtivity or TGT activity after dialysis against assay huffer. A. 'I 'G'I' alone: li . S. crreuisinr [,"'P]t RNA"'" alone (50 pmol): (', T(;T preincuhated with 5'. crrruisinr [:"P]tHNA"" ( 5 0 pmol); I ) . TGT preincuhated with S. crrruisiw [ "PjtRNA"" (50 pmol) in the presence of 1 mM ATI'.

Guanvlyltransferase, Ycast, Histidine tRNA 228.15

A 1 2 3 4 5 6

B -w 1 2

*PPG- I

?

Orlgln- ,

FIG. 5. Characterization of the rtdenylylated tRNA"'" and the differentiall removal of the activating adenylylate from the tRNA by different G-compounds. Aclenvlylation reactions were performed with TGT (2 pg of ATI'-agarose fraction). 0.04 Azrr, purified yeast tRNA"'" and [."P]ATP under standard condition with the addition as indicated. A, autoradiography of a reaction product separation hy gel elect.rophoresis derived from assay with [tr-:"l'jATJ' ( h e I); with addition of 1 mM GTP ( h e 2). 1 mM GDP ( /one 3 1 , and 1 mM G M P ( /one 4 ) ; with no enzyme ( h e 5 ) ; and with addition of [-y-'"P]ATP for [tr-'"P]ATP ( /one 6). H , products of a standard adenylylation reaction were gel purified and sulwequentlv digested with RNase P1 (lone I ) and further treated with snake venom phosphodiesterase (lone 2). The nucleotides were separated hv TLC on polyethyleneimine-cellulose in 1 M ammonium formate (pH 3.5) and visualized hv autoradiography. Appropriate marker nucleotides were cochromat.ographed and visualized under UV light.

denaturing polyacrylamide gel electrophoresis of the phenol- extracted reaction products showed the incorporation of ra- dioactive AMP derived from [n-'"PIATP into tRNA"'", indi- cating the activation of the tRNA by adenylylation (Fig. SA, lane I ) . The control reaction with [-y-'"PIATP did not lead to any incorporation of radioactivity, excluding an activation mechanism by phosphorylation of the tRNA, thus favoring the transfer of the AMP from the adenylylated enzyme to the tRNA (Fig. 5A, lane 6 ) . Interestingly the activation was also observed in the presence of radioactive dATP (data not shown). The 5' end of hte tRNA was analyzed by RNase P1 digestion, which liberated a molecule, which comigrated with A(5')pp(5')G on polyethyleneimine-cellulose T L C (Fig. 5B, lane 1 ) . The further treatment of the obtained oligonucleotide with snake venom phosphodiesterase liberated AMP and served as control reaction for the assumed structure of the RNase P1 digestion product. The two labeled tRNAs (Fig. SA) probably resulted from a difference in the 3' end during tRNA"'" purification of storage, since both activated tRNA'"* molecules contained the same A(5')pp(S')G end.

Phosphodiester Bond Formation with GTP Is the I m t S t e p of tRNA"'" Formation-In the las t s tep of the enzymatic reaction the 3'-hydroxyl of GTP a t tacks the act ivated pG at t h e 5' end of the tRNA with concomitant release of AMP and formation of a new 3'-5'-phosphodiester bond. This reaction sequence was demonstrated hy the disappearane of the acti- vating [:'2P]AMP from the 5' end of tRNA"'" when G T P w a s added (Fig. 5A, lane 2) . Further evidence was provided by the finding that guanosine compounds lacking a 3"hydroxyl were no substrates for the reaction (data not shown). The newly formed phsophodiester bond is 3 '4 ' as shown by the analysis of pGpG being the terminus of the mature tRNA"'" so formed (see accompanying paper).

Guanylyl transferase

F T P

k p 7 Adenylylated Enzyme

k- tRNAH"

I Adenvlvlated tRNA:Enzvme - COmDleX . .

p9 Guanylylated tRNA:Enzyme

n - Guanylylated Guanylyl transferase

FIG. 6. Proposed mechanism for the tRNA""~unn) . l~ l t rnns- tRNA "I'

ferase.

We tested a variety of guanosine derivatives with proper 3"hydroxyl for their abilitv to serve as source for the G- addition reaction. We used the described replacement of the activating ['"PIAMP from the 5' end of the tRNA as meas- urement of the acceptance of the different nucleotides. GTP was found t.o he the most efficient guanosine componnd, totally replacing the AMP on the tRNA (Fig. SA, lnnrp 2) (set a s 100% in Table I ) . dGTP was found almost as efficient as G T P for the guanylylation process (Tahle I ) . While G D P still served for the enzymatic reaction, GMP was accepted verv poorly by the enzyme (Fig. SA, compare lanrs 3 and 4. Tahle I). Other guanosine derivates tested, like dGDP were poor suhstrates for the TGT (Tahle I) . T h e selectivity of the purified enzyme regarding G T P as its source of guanosine makes its role as natural suhstrate very likely. Interestinglv, it was not possible to bind the TGT activitv to any kind of guanosine residue containing affinity chromatography mate- rial (GTP-, GDP-, GMP-, G-agarose with different linker arms), even in the presence of ATP (data not shown). More- over, protein labeling experiments with enzvme immobilized on nitrocellulose membrane as descrihed ahove were per- formed with [n-:"P]GTP without the detection of any laheled protein (Fig. 3, /anP f i ) , indicating that the TGT does not bind G T P per se.

DISCUSSION

The enzymatic mechanism of the S. wrtwisinc TGT ( sum- marized in Fig. 6) shows ohvious homology to the mechanism determined for RNA ligases (Uhlenbeck and Gumport, 1982). The first step is the formation of an enzyme-AMI' complex. As is the case for T4 RNA ligase only dATP suhstitutes for ATP, suggesting that the ATP binding site is highlv specific in its recognition of the adenosine group. Adenylylation of the enzyme generates the hasis for stahle tRNA"" recognition and hinding. Prohahly the dimeric enzyme is needed for tRNA binding as renatured protein bound on nitrocellulose (which could he labeled with ATP) did not hind labeled tRNA"" even in the presence of ATP.' During the next step of the mecha- nism the adenylyl group is transferred from the enzvme to the 5"phosphate of tRNA"". The formation of a S'-T,'-pyro- phosphate linkage activates the 5"phosphate of the tRNA for the subsequent reaction. Finally the 3'-hvdroxyl of G T P attacks the activated 5"phosphate and displaces AMP from the tRNA forming a new phosphodiester bond. The determi- nation of the rate-limiting step for the T G T reaction hy

~~ -

D. .Jahn, rlnpuhlished results.

22836 Guanylyltransferase,

detailed kinetic analysis is one goal for further investigations using recombinant TGT.

The fate of the @- and y-phosphates of the newly incorpo- rated GTP is not clear. Mature tRNAHi“ of yeast and D. melanogster possess a pGpN at the 5’ end (Cooley et al., 1982; Williams et al., 1990), while the mature chicken mitochondrial tRNAH’” has a pppGpN at its 5’ end (Abbe et al., 1990). Whether the removal of the pyrophosphate is catalyzed by the TGT or by a pyrophosphatase is currently under investi- gation. It is pertinent to note that additional modification of the 5”phosphate of the added guanosine compound in D. melanogaster tRNAHi“ has been observed. (Cooley et al., 1982; Williams et al., 1990). The nature of the modification is unknown, but the enzymatic activity is not part of the TGT. Further experiments are needed to decide whether this mod- ification is an essential part of tRNAHis biosynthesis and function.

Acknowledgments-We thank Dr. Dieter Sol1 for support and many discussions. We are indebted to Dr. Martina Jahn for the gift of radioactive labeled E. coli tRNA‘”” and Dr. Stuart Linn for the helpful discussion of the mechanism.

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181,665-672 Xu, Q., Phizicky, E. M., Greer, C. L. and Abelson, J. N. (1990)

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