Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Extracts prepared from tumours of the mouse colon induced by 1,2-dimethylhydrazine were considerably more active in catalysing the methylation of tRNA than were extracts from normal colon. The enhanced activity was observed when both unfractionated ;methyl-deficient' tRNA and purified tRNA preparations from yeast and bacteria were used as substrates for methylation. 2. The methylated bases produced in these reactions were identified. There were no differences between the products of the reaction catalysed by extracts of tumour and normal colon. 3. The increased activity of tRNA methylases was not due to the presence in the extracts of stimulatory or inhibitory molecules of low molecular weight such as polyamines or S-adenosylhomocysteine. 4. Other enzymes concerned with tRNA metabolism (RNA polymerase, ATP-tRNA adenylyltransferase, aminoacyl-tRNA ligases) were also increased in activity in the tumour tissue. 5. The extent of methylation of a limiting amount of tRNA was greater when tumour extracts were compared with controls, but in no case was it possible to achieve a stoicheiometric methylation of the purified tRNA preparations used as substrates, and the tumour extracts were not able to methylate tRNA obtained from normal mouse colon. We conclude that the tumours contained greater activities of tRNA methylases but that there was no evidence for changes in the specificity of these enzymes during neoplastic growth.
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PMID:Further investigation of the increased transfer ribonucleic acid methylase activity in tumours of the mouse colon. 459 40

The universal 3'-terminal CCA sequence of all transfer RNAs (tRNAs) is repaired, and sometimes constructed de novo, by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. This RNA polymerase has no nucleic acid template, yet faithfully builds the CCA sequence one nucleotide at a time using cytidine triphosphate (CTP) and adenosine triphosphate (ATP) as substrates. All previously characterized CCA-adding enzymes from all three kingdoms are single polypeptides with CCA-adding activity. Here, we demonstrate through biochemical and genetic approaches that CCA addition in Aquifex aeolicus requires collaboration between two related polypeptides, one that adds CC and another that adds A.
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PMID:Collaboration between CC- and A-adding enzymes to build and repair the 3'-terminal CCA of tRNA in Aquifex aeolicus. 1170 27

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.
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PMID:Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure. 1459 88

The CCA-adding enzyme ATP(CTP):tRNA nucleotidyltransferase builds and repairs the 3'-terminal CCA sequence of tRNA. Although this unusual RNA polymerase has no nucleic acid template, it can construct the CCA sequence one nucleotide at a time using CTP and ATP as substrates. We found previously that tRNA does not translocate along the enzyme during CCA addition (Yue, D., Weiner, A. M., and Maizels, N. (1998) J. Biol. Chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all three nucleotides (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Intriguingly, the CCA-adding enzyme from the archaeon Sulfolobus shibatae is a homodimer that forms a tetramer upon binding two tRNAs. We therefore asked whether the active form of the S. shibatae enzyme might have two quasi-equivalent active sites, one adding CTP and the other ATP. Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition. We also locate one UV light-induced tRNA cross-link on the enzyme structure and provide evidence suggesting the location of another. Our data rule out shuttling models in which the 3'-end of the tRNA shuttles from one quasi-equivalent active site to another, demonstrate that tRNA-induced tetramerization is not required for CCA addition, and support a role for the tail domain of the enzyme in tRNA binding.
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PMID:A single catalytically active subunit in the multimeric Sulfolobus shibatae CCA-adding enzyme can carry out all three steps of CCA addition. 1526 70

The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.
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PMID:Structural basis for template-independent RNA polymerization. 1529 3

The CCA-adding enzyme, which builds and repairs the 3' terminal CCA sequence of tRNA, is the only RNA polymerase that can synthesize a defined nucleotide sequence without using a nucleic acid template. New cocrystal structures tell us how this remarkable enzyme works.
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PMID:tRNA maturation: RNA polymerization without a nucleic acid template. 1549 78

The universal 3'-terminal CCA sequence of tRNA is built and/or synthesized by the CCA-adding enzyme, CTP:(ATP) tRNA nucleotidyltransferase. This RNA polymerase has no nucleic acid template, but faithfully synthesizes the defined CCA sequence on the 3'-terminus of tRNA at one time, using CTP and ATP as substrates. The mystery of CCA-addition without a nucleic acid template by unique RNA polymerases has long fascinated researchers in the field of RNA enzymology. In this review, the mechanisms of RNA polymerization by the remarkable CCA-adding enzyme and its related enzymes are presented, based on their structural features.
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PMID:Molecular mechanisms of template-independent RNA polymerization by tRNA nucleotidyltransferases. 2459 76

tRNA nucleotidyltransferase adds the invariant CCA-terminus to the tRNA 3'-end, a central step in tRNA maturation. This CCA-adding enzyme is a specialized RNA polymerase that synthesizes the CCA sequence at high fidelity in all kingdoms of life. Recently, an additional function of this enzyme was identified, where it generates a specific degradation tag on structurally unstable tRNAs. This tag consists of an additional repeat of the CCA triplet, leading to a 3'-terminal CCACCA sequence. In order to explain how the enzyme catalyzes this extended polymerization reaction, Kuhn et al. solved a series of co-crystal structures of the CCA-adding enzyme from Archaeoglobus fulgidus in complex with different tRNA substrates. They show that the enzyme forces a bound unstable tRNA to refold the acceptor stem for a second round of CCA-addition, while stable transcripts are robust enough to resist this isomerization. In this review, we discuss how the CCA-adding enzyme uses a simple yet very elegant way to scrutinize its substrates for sufficient structural stability and, consequently, functionality.
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PMID:The CCA-adding enzyme: A central scrutinizer in tRNA quality control. 2617 25

Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.
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PMID:Cold adaptation of tRNA nucleotidyltransferases: A tradeoff in activity, stability and fidelity. 2909 23

tRNAs are important players in the protein synthesis machinery, where they act as adapter molecules for translating the mRNA codons into the corresponding amino acid sequence. In a series of highly conserved maturation steps, the primary transcripts are converted into mature tRNAs. In the amoebozoan Acanthamoeba castellanii, a highly unusual evolution of some of these processing steps was identified that are based on unconventional RNA polymerase activities. In this context, we investigated the synthesis of the 3'-terminal CCA-end that is added posttranscriptionally by a specialized polymerase, the tRNA nucleotidyltransferase (CCA-adding enzyme). The majority of eukaryotic organisms carry only a single gene for a CCA-adding enzyme that acts on both the cytosolic as well as the mitochondrial tRNA pool. In a bioinformatic analysis of the genome of this organism, we identified a surprising multitude of genes for enzymes that contain the active site signature of eukaryotic/eubacterial tRNA nucleotidyltransferases. In vitro activity analyses of these enzymes revealed that two proteins represent bona fide CCA-adding enzymes, one of them carrying an N-terminal sequence corresponding to a putative mitochondrial target signal. The other enzymes have restricted activities and represent CC- and A-adding enzymes, respectively. The A-adding enzyme is of particular interest, as its sequence is closely related to corresponding enzymes from Proteobacteria, indicating a horizontal gene transfer. Interestingly, this unusual diversity of nucleotidyltransferase genes is not restricted to A. castellanii, but is also present in other members of the Acanthamoeba genus, indicating an ancient evolutionary trait.
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PMID:CCA Addition gone wild: Unusual Occurrence and Phylogeny of four different tRNA Nucleotidyltransferases in Acanthamoeba castellanii. 3309 40


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