The acylation of all tRNAs with an amino acid occurs at the terminal ribose of a 3' CCA sequence. The CCA sequence is added to the tRNA precursor by stepwise nucleotide addition performed by a single enzyme that is ubiquitous in all living organisms. Although the enzyme has the option of releasing the product after each addition, it prefers to stay bound to the product and proceed with the next addition .
cca-adding enzyme, trnt1, cca enzyme, cca1p, atp(ctp):trna nucleotidyltransferase, trna-nt, class i cca-adding enzyme, ccase, afcca, ctp(atp):trna nucleotidyltransferase, more
The acylation of all tRNAs with an amino acid occurs at the terminal ribose of a 3' CCA sequence. The CCA sequence is added to the tRNA precursor by stepwise nucleotide addition performed by a single enzyme that is ubiquitous in all living organisms. Although the enzyme has the option of releasing the product after each addition, it prefers to stay bound to the product and proceed with the next addition [5].
the 30-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis
tRNA does not rotate or translocate during C74 addition. A single flexible beta-turn orchestrates consecutive addition of all three nucleotides without significant movement of the tRNA on the enzyme surface
tRNA minihelices, which contain only the acceptor stem and TPsiC stem-loop, are also efficiently subjected to CCACCA addition when they have guanosines at the first and second positions as well as a destabilized acceptor stem by virtue of mismatches and G-U wobbles
CCA-adding enzymes recognize tRNA and tRNA-like structures as substrates, select and discriminate the correct nucleotides CTP and ATP against UTP and GTP, and, after incorporation of two C residues, the nucleotide specificity has to switch towards ATP without the help of a nucleic acid template. The enzymes have to stop polymerization exactly after three positions and recognize partial CCA-ends and add only the missing residues for completion, instead of stubbornly adding CCA-ends to their substrates, overview
CCA-adding enzymes recognize tRNA and tRNA-like structures as substrates, select and discriminate the correct nucleotides CTP and ATP against UTP and GTP, and, after incorporation of two C residues, the nucleotide specificity has to switch towards ATP without the help of a nucleic acid template. The enzymes have to stop polymerization exactly after three positions and recognize partial CCA-ends and add only the missing residues for completion, instead of stubbornly adding CCA-ends to their substrates, overview
essentially required, two metal ions are coordinated by highly conserved carboxylates and fulfill specific roles in catalyzing the reaction. Metal ion A activates the 3'-hydroxyl group of the primer for a nucleophilic in-line attack on the alpha-phosphate of the incoming NTP, while metal ion B promotes the leaving of the diphosphate group that is released during this reaction
two metal ions are bound to the two catalytically important carboxylates. The first metal ion deprotonates the 3'-OH group of the tRNA primer and activates the resulting 3'-O for an attack at the a-phosphate of the incoming nucleotide. The second metal ion stabilizes the triphosphate moiety of the NTP and facilitates the leaving of the pyrophosphate group, overview
whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates
a class I CCA-adding enzyme. CCA-adding enzymes are essential RNA polymerases that emerged twice in evolution leading to different structural characteristics and unusual mechanistic solutions for an error-free and sequence-specific CCA polymerization reaction. The catalytic cleft is formed by the head, neck, and body domains. Evolution of class I and class II CCA-adding enzymes as well as poly(A) polymerases, overview
diphosphorolysis 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
CCA-adding enzymes represent vital components of the cell's tRNA maturation and maintenance system. The CCA end, added to the tRNA by the CCA tRNA nucleotidyltransferase, is the site of aminoacylation, and aminoacyl tRNA synthetases fuse the individual amino acids to the ribose moiety of the terminal A residue. Second, the CCA terminus is required for the correct positioning of the aminoacyl-tRNA in the ribosome's A- and P-site in order to guarantee an efficient peptidyl transfer reaction. The CCA-adding enzyme represents an essential activity in the majority of organisms
structure-function relationship, overview. The enzyme binds the tRNA top half in the correct orientation for CCA-addition in a cleft, the tRNA acceptor stem interacts with a highly conserved long alpha-helical element in an almost parallel orientation. In the position of nucleotide addition, the 3'-end is bound to the active site located in the enzyme's head domain, while the T loop of the tRNA contacts the tail domain. The bound tRNA substrate remains fixed at its binding site in the enzyme during the complete nucleotide incorporation process
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, reaction mechanism of CCA addition, overview
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
cocrystal structures of the enzyme complexed with both a tRNA mimic and nucleoside triphosphates under catalytically active conditions. The structures suggest that adenosine 5'-monophosphate is incorporated onto the A76 position of the tRNA via a carboxylate-assisted, one-metal-ion mechanism with aspartate 110 functioning as a general base. The discrimination against incorporation of cytidine 5'-triphosphate at position 76 arises from improper placement of the a phosphate of the incoming CTP, which results from the interaction of C with arginine 224 and prevents the nucleophilic attack by the 3' hydroxyl group of cytidine75
cocrystallisation of enzyme complexes with nine distinct tRNA minihelices. All of the binary and ternary complex crystals belong to the space group P4(3)2(1)2, and contain one complex molecule in the asymmetric unit. Crystal structures are solved at 2.5 to 3.05 A resolutions by molecular replacement
in complex with a human MenBeta minihelix. The unstable minihelix is bound between the enzymes catalytic center, comprised of the head and neck domains, and its tail domain. The minihelix perfectly mimics full-length tRNA with its acceptor and TPsiC stems folding into a continuous A-type RNA helix. The TPsiC loop is in the same conformation as in full-length tRNA
kcat /Km for C74 addition is 28% of wild-type value, kcat /Km for C75 addition is 33% of wild-type value, kcat /Km for A76 addition is 1% of wild-type value
kcat /Km for C74 addition is 94% of wild-type value, kcat /Km for C75 addition is 15% of wild-type value, kcat /Km for A76 addition is 129% of wild-type value
kcat /Km for C74 addition is 3% of wild-type value, kcat /Km for C75 addition is 1% of wild-type value, kcat /Km for A76 addition is 1% of wild-type value
kcat /Km for C74 addition is 36% of wild-type value, kcat /Km for C75 addition is 67% of wild-type value, kcat /Km for A76 addition is 92% of wild-type value