From the Department of Biochemistry and Biophysics,
University of Rochester School of Medicine, Rochester, New York
14642, the § Department of Chemistry, University of
Rochester, Rochester, New York 14627, and the ¶ Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
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ABSTRACT |
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The last step of tRNA splicing in yeast is
catalyzed by Tpt1 protein, which transfers the 2'-phosphate from
ligated tRNA to NAD to produce ADP-ribose 1"-2"-cyclic phosphate
(Appr>p). Structural and functional TPT1 homologs are
found widely in eukaryotes and, surprisingly, also in Escherichia
coli, which does not have this class of tRNA splicing. To
understand the possible roles of the Tpt1 enzymes as well as the
unusual use of NAD, the reaction mechanism of the E. coli
homolog KptA was investigated. We show here that KptA protein removes
the 2'-phosphate from RNA via an intermediate in which the phosphate is
ADP-ribosylated followed by a presumed transesterification to release
the RNA and generate Appr>p. The intermediate was characterized by
analysis of its components and their linkages, using various labeled
substrates and cofactors. Because the yeast and mouse Tpt1 proteins,
like KptA protein, can catalyze the conversion of the KptA-generated
intermediate to both product and the original substrate, these enzymes
likely use the same reaction mechanism. Step 1 of this reaction is
strikingly similar to the ADP-ribosylation of proteins catalyzed by a
number of bacterial toxins.
tRNA introns occur widely in Eukarya and Archaea (1, 2). Splicing
in these organisms is initiated by a highly conserved endonuclease that
excises the intron (3-5), followed by joining of the two
half-molecules by one of two different ligases (6-13). In the yeast
Saccharomyces cerevisiae, in which the process is best
studied, ligation occurs by a four-step reaction, producing a splice
junction with a 2'-phosphate (14, 15).
A single essential gene (TPT1) encodes the
2'-phosphotransferase responsible for removal of the splice junction
2'-phosphate from ligated tRNA (16, 17). This reaction is unusual
because the 2'-phosphate is transferred to NAD (18), producing mature tRNA and ADP-ribose 1"-2" cyclic phosphate
(Appr>p)1 (19). The
TPT1 gene product is involved in this step in
vivo because a conditional yeast tpt1 mutant, when
depleted for the gene product, accumulates at least eight ligated tRNA
species bearing a splice junction 2'-phosphate (17). This presumably is
the essential function of Tpt1 protein. Examination of four of these
tRNAs demonstrated that they are also undermodified specifically at the
splice junction residue (17).
The yeast Tpt1 protein is part of a family of functional
2'-phosphotransferases found in Eukarya (Schizosaccharomyces
pombe, Candida albicans, Arabidopsis
thaliana and Mus musculus), and Eubacteria
(Escherichia coli), with other likely members in
another bacterial species and several Archaea (46). Expression of the eukaryotic phosphotransferase genes and the E. coli gene
(kptA) complements a yeast tpt1 mutant, and the
corresponding proteins catalyze the same reaction as the yeast protein,
producing Appr>p from ligated tRNA and NAD. The widespread occurrence
of 2'-phosphotransferases in Eukarya is consistent with the ubiquitous
presence of intron-containing tRNAs and ligases that generate 2'-
phosphorylated substrates in eukaryotes. However, a functional
2'-phosphotransferase was not anticipated in E. coli and
bacteria in general because they do not have the corresponding ligase
or this class of tRNA splicing. Bacteria only have the distinctively
different group I and group II self-splicing introns (20-22).
Moreover, phylogenetic analysis indicates that the bacterial gene is
ancient. It is unclear what the function of the E. coli
protein might be. One possible role for the E. coli protein
is the catalysis of a related chemical reaction, which might also be
catalyzed by other Tpt1 homologs.
To learn the spectrum of reactions that could be catalyzed by the Tpt1
enzyme family and to understand the unusual use of NAD in the
phosphotransferase reaction, we undertook to learn the reaction
mechanism. Two mechanistic pathways that could account for the
formation of Appr>p are illustrated in Fig.
1. In mechanism A (19), the first step is
a phosphoryl transfer in which the 2'-phosphate at the splice junction
is transferred to the 2"-hydroxyl of the NMN ribose of NAD, releasing
the dephosphorylated RNA. This is followed by cyclization of the
phosphate to the 1" position of NAD, with concomitant release of
nicotinamide. In mechanism B, the phosphodiester bond is formed in step
1 by formation of a covalent bond between the 2'-phosphate of tRNA and
the 1" position of NAD, releasing nicotinamide. The second step is then
a simple transesterification reaction, where the 2"-hydroxyl of NAD
displaces the tRNA 2'-OH in the phosphodiester linkage. In both
mechanisms the energy for phosphodiester bond formation is derived from
the hydrolysis of the C-N glycosidic bond joining ribose and
nicotinamide.
INTRODUCTION
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Abstract
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Fig. 1.
Two plausible reaction mechanisms for
2'-phosphotransferase. Schematics of two possible reaction
mechanisms of the 2'-phosphotransferase are presented, each of which
could account for the transfer of the 2'-phosphate from ligated tRNA to
NAD to produce Appr>p. Nic, nicotinamide. Material
presented in the text supports mechanism B.
We provide evidence here that mechanism B is correct, based on the
identification and characterization of a reaction intermediate generated by the E. coli 2'-phosphotransferase, KptA.
Further, we show that the bacterial mechanism is conserved for yeast
(Tpt1), and mouse (mTpt1) phosphotransferase proteins. Step 1 of
mechanism B is related to a class of ADP-ribosylating reactions found
in a variety of bacterial toxins and other ADP-ribosyl transferases.
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EXPERIMENTAL PROCEDURES |
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Labeling of Substrates--
ApAPpA was
5'-phosphorylated in 20-µl reactions containing 100 µM
ApAPpA, 5 µCi of [-32P]ATP (3,000 Ci/mmol), and 1 unit of polynucleotide kinase (3'-phosphatase-free) in
buffer (Boehringer Mannheim). Reactions were incubated for 30 min at
37 °C and applied to silica thin layer chromatography plates (J. T.
Baker), and products were separated in buffer containing n-propyl alcohol:NH4OH:H2O, 55:35:10
v/v/v and eluted in H2O. [32P-adenylate]NAD was prepared from
[
-32P]ATP (3,000 Ci/mmol) and NMN as described (23).
Reaction mixtures were treated subsequently with 0.01 unit of calf
intestinal phosphatase for 30 min, phenol extracted, and applied to
silica thin layer chromatography plates; products were resolved in
buffer containing ethanol and 1 M NH4OAc, pH
7.2, 7:3 v/v, eluted in water, and dried. [3H]NAD was
prepared as described by Little (24). p*-intermediate was made from
12-30 fmol of p*ApAPpA (or
p*ApApAPPOCH3) in the presence of 5 mM NAD, and products were purified by chromatography on
silica thin layer plates developed in buffer containing ethanol and 1 M NH4OAc, pH 7.2, 7:4.5 v/v, followed by
elution in water and drying. Although this procedure yields intermediate that is partially contaminated with some RNA substrate and
product (because of the poor separation on these plates), the eluted
intermediate lacks NAD and is active (see "Results"). Ap*pN
intermediate was purified the same way, after synthesis as described in
Fig. 6.
2'-Phosphotransferase-- Phosphotransferase activity was assayed for 30 min as described (16) at 30 °C for yeast (Tpt1) and mouse (mTpt1) phosphotransferases and at 37 °C for KptA from E. coli. Analysis was by TLC on polyethyleneimine (PEI)-cellulose plates developed in 2 M sodium formate, pH 3.5, unless otherwise stated.
Proteins--
KptA and mTpt1 were prepared from extracts of
E. coli cells expressing the protein by Blue Sepharose
column chromatography.2 Yeast Tpt1 was prepared by M. Steiger as a Tpt1-His6 fusion protein and was purified from
E. coli extracts by nickel column affinity chromatography.
Yeast cyclic phosphodiesterase protein has been described (23). Calf
intestinal alkaline phosphatase (Boehringer Mannheim) was diluted to
0.01 units/µl in 300 mM NaCl, 1 mM
MgCl2, and 0.1 mM ZnCl2, and 1 µl
was added for 30 min at 37 °C. Pyrophosphatase reactions contained
10 mM Tris-HCl, pH 9, 10 mM MgCl2,
1 µg of carrier RNA, and 0.1-1 unit of nucleotide pyrophosphatase
(type II: Crotalus adamanteus; Sigma) in 10 µl of buffer
and were incubated at 37 °C for 60 min.
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RESULTS |
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The E. coli 2'-Phosphotransferase Protein Produces a Reaction Intermediate-- To examine the mechanism of the E. coli 2'-phosphotransferase (KptA protein), we used a 5'-32P end-labeled synthetic RNA trimer containing an internal 2'-phosphate (p*ApAPpA, where the * indicates the position of the labeled phosphate). This substrate and several other synthetic RNAs have been characterized extensively by NMR and analysis with nucleases and phosphatases2 ; a similar substrate has been used with the yeast enzyme (19).
As shown in Fig. 2, titration of p*ApAPpA with increasing amounts of the E. coli enzyme in the presence of NAD produces both the expected product (p*ApApA) and in some cases, an additional product. This additional product (designated p*-intermediate) is most prominent in lane f, in which the amount of substrate and final product are roughly equal. It seems unlikely that this additional product is the result of a contaminating activity in the enzyme preparation because the amount of the product decreases as more enzyme is added to the reaction (lanes h-j). Rather, the additional product appears to be an intermediate because there is less of it at both lower and higher concentrations of enzyme.
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If there is an intermediate, it should appear before product forms and disappear as product accumulates. This is demonstrated in Fig. 3, which displays the results of a time course of the reaction at several concentrations of the E. coli phosphotransferase. In the presence of 1 unit of enzyme activity (panel C), it is clear that at the early time points (up to 40 min), there is substantially more of the spot labeled p*-intermediate than of product, and at later time points (after 80 min), there is substantially less p*-intermediate and correspondingly more product. This is quantitated in Fig. 3 (panel F). With less KptA protein (panels D and G), it is even more obvious that the intermediate forms first. With more enzyme (panels B and E), it is clear that p*-intermediate is depleted quantitatively at the end of the reaction. Thus, the spot labeled p*-intermediate has the kinetic properties expected of a reaction intermediate. In addition, this spot reaches a high concentration during the reaction (27%, see panels C and F), indicating that a large percentage of the substrate goes through the intermediate state to become product.
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If the spot labeled p*-intermediate is an intermediate, it should also be converted to product by KptA protein. To test this, p*-intermediate was isolated from silica TLC plates. As shown in Fig. 4 (lane e) this yields p*intermediate that is slightly contaminated with substrate RNA and product. As shown in lane g, all of the intermediate is converted to product in the presence of KptA and NAD. Thus, we conclude that the spot labeled p*-intermediate has both properties expected of a reaction intermediate: it appears and disappears at times expected of an intermediate, and it can be converted to product. Because the RNA is labeled, these results also demonstrate directly that RNA is part of the intermediate, as predicted by mechanism B (Fig. 1).
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NAD Is a Component of the Intermediate-- Two lines of evidence support the claim that NAD is part of the intermediate formed with labeled RNA. First, catalysis of step 2 of the reaction with isolated p*-intermediate does not require addition of NAD, indicating that NAD is already present in the intermediate (Fig. 4, compare lanes f and g with lane e). We note that there is little, if any, free NAD in this preparation of intermediate because the small amount of contaminating substrate (p*ApAPpA) in the intermediate (lane e) requires added NAD to be converted to product in the presence of KptA (compare lanes f and g). Second, an intermediate with altered mobility forms with chemical derivatives of NAD. This is illustrated in Fig. 5, where we have compared reactions with either NAD or NGD (nicotinamide guanine dinucleotide). It is clear that an intermediate of different mobility is formed with NGD (compare lanes c-e with lanes m and n).
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In addition, an intermediate is formed with labeled NAD. To show this we used [32P-adenylate]NAD (Ap*pN) in a phosphotransferase reaction with unlabeled RNA (ApAPpA). As shown in Fig. 6 (lanes c-e), two products are formed. One of these comigrates with Ap*pr>p and increases with more protein in the reaction. The other is designated Ap*pN intermediate because, like the p*-intermediate, its levels are maximal when roughly half of the final amount of product is formed (lane d). As with the p*-intermediate, the isolated Ap*pN intermediate is efficiently converted to product, in this case to Ap*pr>p, with or without NAD (Fig. 7, compare lane a with lanes c and d). As expected for Ap*pr>p, the product is resistant to phosphatase (lane e) and is a substrate for a yeast cyclic phosphodiesterase that converts Ap*pr>p to Ap*pr1"p (lane f) (23).
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The p*-intermediate formed with labeled RNA and the Ap*pN intermediate formed with labeled NAD are likely the same, for two reasons. First, the change in the mobility of the Ap*pN intermediate formed with ApAPpA (Figs. 6 and 7) is consistent with its reduced charge compared with the intermediate formed with labeled RNA (p*ApAPpA), which has an extra phosphate. Second, we have demonstrated comigration of the RNA-labeled p*-intermediate with that of an intermediate formed with Ap*pN and unlabeled pApAPpA (data not shown). Moreover, the different conditions required to detect the Ap*pN intermediate and the p*-intermediate are explained readily by the kinetic parameters of the enzyme. We have found that the intermediate forms much less efficiently at low concentrations of NAD (data not shown). Therefore, additional unlabeled NAD was used in the reaction with labeled NAD, which requires both extra substrate RNA (to convert a reasonable amount of the label to product) and correspondingly more protein to catalyze the reaction. Nonetheless, the amount of protein required to form the intermediate in each case conforms very closely to that expected from the kinetic parameters.2
The fact that both RNA and NAD comprise the intermediate eliminates mechanism A as the catalytic pathway for the phosphotransfer reaction (Fig. 1). The results are consistent with mechanism B, and the experiments described below, which further probe the structure of the intermediate, lend support to this conclusion.
The RNA 2'-Phosphate Is Linked to the NMN Portion of NAD-- To determine if the 2'-phosphate of the RNA is involved in the NAD-RNA linkage, as predicted by mechanism B, we treated both p*-intermediate and the Ap*pN intermediate with phosphatase. As shown in Fig. 7 (lane b), there is no change in the mobility of the Ap*pN intermediate after phosphatase treatment (although a minor contaminant is sensitive), indicating that the 2'-phosphate is involved in the linkage. By contrast, treatment of p*-intermediate results in the formation of inorganic phosphate (Fig. 4, lane h), demonstrating that the 5'-32P-labeled phosphate of the RNA substrate is not involved in the linkage.
As predicted by mechanism B, the 2'-phosphate is linked to the NMN moiety of NAD. To show this, we treated the p*-intermediates formed with NAD and NGD (see Fig. 5) with pyrophosphatase to remove the AMP (GMP) portion of the molecule. Because this removes the distinguishing portion of NAD and NGD, the position of RNA attachment to NAD can be determined simply by investigation of the migration properties of the released labeled products. Since the pyrophosphatase digestion products comigrated in our TLC system, this indicates that the attachment is to NMN (data not shown).
Nicotinamide Is Released during Intermediate Formation--
Two
approaches were used to demonstrate that nicotinamide is released as
intermediate is formed, as predicted in mechanism B. First, we
demonstrated that phosphotransferase can catalyze the reversal of step
1 in the presence of nicotinamide (Fig.
8, panel A). Whereas all of
the intermediate is converted to product from isolated p*-intermediate
in the presence of KptA protein (lane c), addition of
nicotinamide at concentrations greater than 0.1 mM results
in formation of the original substrate (p*ApAppA)
(lanes e and f). Quantitation at 10 mM nicotinamide indicates that there is conversion of the
intermediate to 70% substrate and
30% product. Similarly, the
Ap*pN intermediate can be converted to the original substrate (Ap*pN)
with excess nicotinamide (Fig. 8, panel B, lanes
h and i). Because nicotinamide addition reverses the
phosphotransferase reaction, we presume it is not part of the
intermediate. Consistent with this, we found that the addition of
thionicotinamide to Ap*pN intermediate results in the formation of
thionicotinamide adenine dinucleotide (data not shown).
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Second, we showed directly that nicotinamide release occurs well before formation of product (data not shown). This was accomplished by conducting a time course of the release of [3H]nicotinamide from [3H]NAD. To correlate nicotinamide release with reaction progress, we used a second labeled substrate, p*ApAPpA, in the same reaction, at comparable concentrations (and at similar intensity after autoradiography). The [3H]nicotinamide was clearly formed (10% at 10 min) well before formation of product p*ApApA (7% at 40 min). Presumably nicotinamide release occurs during intermediate formation.
Because nicotinamide is absent from the intermediate and the 2'-phosphate is engaged in the linkage to NAD, the most reasonable structure of the intermediate has the RNA 2'-phosphate linked at the 1" position. If so, the adjacent 2"- and 3"-hydroxyls should be sensitive to periodate. To test this, we used p*ApApAPPOCH3 as substrate because it has no vicinal hydroxyls on its riboses.2 After forming the intermediate with AppN, we treated it with pyrophosphatase to release the AMP moiety (Fig. 9, lane c); this removed the vicinal hydroxyls on the ribose of AMP. As shown in lane d, the remaining material is periodate-sensitive, which could only occur if the 2"-hydroxyl is free. We conclude that step 1 involves attachment of the RNA 2'-phosphate to the 1" position as depicted in mechanism B of Fig. 1. (The alternative explanation, that the RNA 2'-phosphate is attached to the 3" position of ADP-ribose, would make the reaction too complicated to form the final product Appr>p). It follows that step 2 is then a simple transesterification reaction, resulting in cyclization of the phosphate at the 2" position of the ribose.
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Yeast and Mouse 2'-Phosphotransferases Can Catalyze Step 2 of the Reaction and the Reverse of Step 1-- Because the E. coli protein is part of a highly conserved family that includes the yeast (Tpt1) and mouse (mTpt1) phosphotransferases, the mechanism of the reaction should be similarly conserved. Three lines of evidence support this claim. First, in the presence of high concentrations of NAD, a small amount of intermediate is formed with the yeast and mouse phosphotransferases, which comigrates with the intermediate formed with KptA protein (data not shown). Second, both Tpt1 and mTpt1 proteins can catalyze the conversion of the E. coli-produced intermediate to product (Fig. 10, lanes b and c; data not shown for mouse). Third, both yeast and mouse can catalyze the reverse of step 1: the KptA intermediate is converted to the original substrate when an excess of nicotinamide is added to the reaction (Fig. 11, lanes c and d). Because the yeast and mouse phosphotransferases can catalyze both the forward and the reverse reactions from the intermediate, they must be able to catalyze the reaction by the same mechanism.
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DISCUSSION |
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Isolation and characterization of a reaction intermediate provides
evidence that removal of the splice junction 2'-phosphate from ligated
tRNA during the last step of splicing involves transient ADP-ribosylation of that phosphate. Individual labeling of the RNA
substrate, the adenylic acid phosphate of NAD, and nicotinamide indicates that both RNA and NAD are part of the intermediate and that
nicotinamide is absent. Consistent with this, the intermediate can be
converted to product in the absence of both RNA substrate and NAD and
is converted back to substrate in the presence of excess nicotinamide.
As depicted in mechanism B of Fig. 1, these results suggest that step 1 of the reaction is a nucleophilic attack of the RNA 2'-phosphate on the
1" position of NAD. Evidence for this attachment in the intermediate
includes the phosphatase resistance of the RNA 2'-phosphate after
intermediate formation and the periodate sensitivity of the ribose that
released nicotinamide. This attachment is also consistent with
energetic requirements: release of nicotinamide during step 1 would
provide sufficient energy for phosphodiester bond formation between the
RNA 2'-phosphate and the NMN ribose of NAD because the free energy of
hydrolysis of the N-glycosidic bond of NAD is 8.2 kcal/mol
(25, 26). Step 2 is then a transesterification reaction, resulting in
cyclization of the phosphate to the 2" position of ADP-ribose and the
release of RNA. Our evidence also indicates that the yeast and mouse
phosphotransferases use the same mechanism as the E. coli
protein because both Tpt1 and mTpt1 proteins are able to catalyze the
conversion of the isolated E. coli intermediate both forward
to product and backward to substrate. Presumably, other members of this
widespread family of 2'-phosphotransferase enzymes catalyze this
reaction in the same way. Although we have not yet demonstrated that
the intermediate forms with tRNA, it seems probable that the mechanism
is the same with this substrate.
The intermediate described here is one of a surprisingly large number of chemical alterations that are formed one base 3' of the anticodon (the hypermodified position) in tRNAs that are spliced. During the first two steps of splicing, this nucleotide bears a 2'-3'-cyclic phosphate (4, 27), and then a 2'-phosphate (28) before the half-molecules are joined to generate a splice junction phosphodiester with a 2'-phosphate (28). As described here, the 2'-phosphate is then modified by addition of an ADP-ribose adduct before removal of the phosphate as Appr>p (19) and formation of tRNA with a splice junction 2'-OH (17). Finally, the base of this residue can be modified efficiently to its hypermodified state (17). The significance of the multiple steps at this one residue is unclear; perhaps they occur to ensure that immature tRNA is not used inappropriately in the cell (1).
Step 1 of the reaction is strikingly similar to the ADP-ribosylation catalyzed by a number of well studied bacterial toxins that exert their effects by modification of protein synthesis factors, structural proteins, or signal transduction proteins. In the case of the 2'-phosphotransferase, the nucleophile is the 2'-phosphate of the RNA. By contrast, the nucleophile for the toxins can be a variety of amino acid residues: diphthamide (modified histidine) for Pseudomonas exotoxin A and diphtheria toxin (29, 30); arginine for choleragen toxin, Clostridium botulinum C2, and dintrogenase reductase ADP-ribosyl transferase (31-34); cysteine for pertussis toxin (35); and asparagine for C1 botulinum toxin (36). We noted previously that the family of 2'- phosphotransferases shares sequence similarity with the NAD binding site of diphtheria toxin and Pseudomonas exotoxin A (16). Two other reactions that are similar to step 1 include the first step of poly(A)DP-ribose synthetase, in which glutamate acts as the nucleophile (37), and NAD glycohydrolases, which hydrolyze NAD to ADP-ribose and nicotinamide (38, 39).
In the second step, KptA presumably catalyzes a transesterification reaction, in which the 2"-O of ADP-ribose displaces the 2'-O of tRNA in the phosphodiester bond, generating a cyclic phosphate. Step 2 is comparable to the first step of a class of RNases where hydrolysis of the phosphodiester bond is catalyzed by attack of an adjacent hydroxyl to form a cyclic phosphate intermediate (40). An identical reaction occurs in tRNA splicing, in which the endonuclease generates a 2'-3'-cyclic phosphate terminus on the 5'-half molecule during hydrolysis to excise the intron (27). This step is also similar to the transesterification reactions of RNA-catalyzed self-splicing (41).
It is possible that KptA protein functions in E. coli as an
ADP-ribosylating enzyme. Such a role would be consistent with the
observation that the bacterial protein catalyzes the second step of the
reaction poorly compared with the yeast and mouse phosphotransferases.
This is why it is difficult to observe the intermediate with the yeast
and mouse proteins. Furthermore, we argued previously that for an
ancient bacterial protein still to be functional in yeast, it must have
retained a high degree of substrate specificity and chemical
reactivity, implying that its role is related to its catalytic function
(46). Several cases of ADP-ribosylation of endogenous proteins have
been reported in bacteria (E. coli, Pseudomonas
maltophilia and Streptomycin triseus) (42-44), as well
as the well studied regulatory ADP-ribosylation of dintrogenase
reductase which occurs in the photosynthetic bacterium, Rhodospirillum rubrum (45). The E. coli protein
might ADP-ribosylate a protein, as do the other proteins with this
activity, or perhaps RNAs (as described here) or small molecules. Such
a role might also be conserved in the eukaryotic
2'-phosphotransferases. Alternatively, there may be some other
metabolite or small molecule that requires removal of its phosphate in
E. coli, either to effect dephosphorylation or to produce
Appr>p.
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ACKNOWLEDGEMENTS |
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We thank Michelle Steiger for yeast Tpt1 protein, and Mark Dumont, Elizabeth Grayhack, and Michelle Steiger for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM52347 (to E. M. P.) and GM22939 (to D. H. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel:
716-275-7268; Fax: 716-271-2683; E-mail: eric_phizicky{at}urmc.rochester.edu.
The abbreviations used are: Appr>p, 1"-2"-cyclic phosphate; AppN, NAD; PEI, polyethyleneimine; p*ApApA, p*ApAPpA, pApAPpA, p*ApApAPPOCH3, superscript P is a 2-phosphate.
2 M. A. Steiger, R. Kierzek, D. H. Turner, and E. M. Phizicky, unpublished observations.
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REFERENCES |
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