Pyrophosphorolysis of CCA addition: implication for fidelity

In nucleic acid polymerization reaction, pyrophosphorolysis is the reversal of nucleotide addition, in which the terminal nucleotide is excised in the presence of inorganic pyrophosphate (PPi). The CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3′ end without using a nucleic acid template. To better understand the reaction mechanism of CCA addition, we tested pyrophosphorolysis of CCA enzymes, which are divided into two structurally distinct classes. Here, we show that only class II CCA enzymes catalyze pyrophosphorolysis and that the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Pyrophosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions. Importantly, pyrophosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3′ end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. Measurement of kinetic parameters of the class II Escherichia coli CCA enzyme reveals that the enzyme catalyzes pyrophosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to PPi relative to NTP, suggesting a mechanism in which PPi is rapidly released after each nucleotide addition as a driving force to promote the forward synthesis of CCA.     

Poly(C) synthesis by class I and class II CCA-adding enzymes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.     

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.     

A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.     

Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.     

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

The CCA-adding enzyme repairs the 3''-terminal CCA sequence of all tRNAs. To determine how the enzyme recognizes tRNA, we probed critical contacts between tRNA substrates and the archaeal Sulfolobus shibatae class I and the eubacterial Escherichia coli class II CCA-adding enzymes. Both CTP addition to tRNA-C and ATP addition to tRNA-CC were dramatically inhibited by alkylation of the same tRNA phosphates in the acceptor stem and TPsiC stem-loop. Both enzymes also protected the same tRNA phosphates in tRNA-C and tRNA-CC. Thus the tRNA substrate must remain fixed on the enzyme surface during CA addition. Indeed, tRNA-C cross-linked to the S. shibatae enzyme remains fully active for addition of CTP and ATP. We propose that the growing 3''-terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single CTP binding site. The ATP binding site would then be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would be a dynamic ribonucleoprotein structure.     

tRNA-nucleotidyltransferases: Highly unusual RNA polymerases with vital functions

tRNA-nucleotidyltransferases are fascinating and unusual RNA polymerases responsible for the synthesis of the nucleotide triplet CCA at the 3′-terminus of tRNAs. As this CCA end represents an essential functional element for aminoacylation and translation, these polymerases (CCA-adding enzymes) are of vital importance in all organisms. With a possible origin of ancient telomerase-like activity, the CCA-adding enzymes obviously emerged twice during evolution, leading to structurally different, but functionally identical enzymes. The evolution as well as the unique polymerization features of these interesting proteins will be discussed in this review.     

A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme.

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] catalyzes the addition and regeneration of the 3'-terminal CCA sequence of tRNAs. We show that the CCA-adding enzyme will specifically add a CCA terminus to synthetic full-length tDNA and to DNA oligonucleotides corresponding to the "top half" of tRNA-the acceptor stem and TpsiC stem-loop of tRNA. CCA addition to the top half tDNA minihelices requires a 2' as well as a 3' OH at the 3' terminus of the tDNA. Addition also depends on the length of the base paired stem, and is facilitated by, but is not dependent upon, the presence of a TpsiC loop. These results provide further evidence for independent functions of the top and bottom halves of tRNA, and support the hypothesis that these two structurally distinct and functionally independent domains evolved independently.     

U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase).

The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.     

Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure

CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes.     

Unusual synthesis by the Escherichia coli CCA-adding enzyme

The tRNA 3' end contains the conserved CCA sequence at the 74-76 positions. The CCA sequence is synthesized and maintained by the CCA-adding enzymes. The specificity of the Escherichia coli enzyme at each of the 74-76 positions was investigated using synthetic minihelix substrates that contain permuted 3' ends. Results here indicate that the enzyme has the ability to synthesize unusual 3' ends. When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs. Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not. In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme. However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA. The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs. Thus, the E. coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP. These features led to a model of CCA synthesis that is independent of a nucleic acid template. The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.     

Closely related CC- and A-adding enzymes collaborate to construct and repair the 3'-terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

The 3'-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CC-adding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334-1336). Here we show that in Synechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC- and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time.     

A tRNA's fate is decided at its 3′ end: Collaborative actions of CCA-adding enzyme and RNases involved in tRNA processing and degradation

tRNAs are key players in translation and are additionally involved in a wide range of distinct cellular processes. The vital importance of tRNAs becomes evident in numerous diseases that are linked to defective tRNA molecules. It is therefore not surprising that the structural intactness of tRNAs is continuously scrutinized and defective tRNAs are eliminated. In this process, erroneous tRNAs are tagged with single-stranded RNA sequences that are recognized by degrading exonucleases. Recent discoveries have revealed that the CCA-adding enzyme – actually responsible for the de novo synthesis of the 3′-CCA end – plays an indispensable role in tRNA quality control by incorporating a second CCA triplet that is recognized as a degradation tag. In this review, we give an update on the latest findings regarding tRNA quality control that turns out to represent an interplay of the CCA-adding enzyme and RNases involved in tRNA degradation and maturation. In particular, the RNase-induced turnover of the CCA end is now recognized as a trigger for the CCA-adding enzyme to repeatedly scrutinize the structural intactness of a tRNA. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.     

Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes

CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.     

A comparative analysis of two conserved motifs in bacterial poly(A) polymerase and CCA-adding enzyme

Showing a high sequence similarity, the evolutionary closely related bacterial poly(A) polymerases (PAP) and CCA-adding enzymes catalyze quite different reactions—PAP adds poly(A) tails to RNA 3′-ends, while CCA-adding enzymes synthesize the sequence CCA at the 3′-terminus of tRNAs. Here, two highly conserved structural elements of the corresponding Escherichia coli enzymes were characterized. The first element is a set of amino acids that was identified in CCA-adding enzymes as a template region determining the enzymes' specificity for CTP and ATP. The same element is also present in PAP, where it confers ATP specificity. The second investigated region corresponds to a flexible loop in CCA-adding enzymes and is involved in the incorporation of the terminal A-residue. Although, PAP seems to carry a similar flexible region, the functional relevance of this element in PAP is not known. The presented results show that the template region has an essential function in both enzymes, while the second element is surprisingly dispensable in PAP. The data support the idea that the bacterial PAP descends from CCA-adding enzymes and still carries some of the structural elements required for CCA-addition as an evolutionary relic and is now fixed in a conformation specific for A-addition.