Archaeal CCA-adding Enzymes

Cho, HyunDae D.; Verlinde, Christophe L. M. J.; Weiner, Alan M.

doi:10.1074/jbc.m412594200

Cited by 24 publications

(38 citation statements)

References 52 publications

(110 reference statements)

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“…Both enzymes contain β-turn structures close to the active site and sequence motifs located on these β-turns seem to be specific for CCA or poly(A) addition [38,39]. It was also demonstrated that in CCAtrs the tRNA precursor substrate does not translocate during CCA synthesis: If the tRNA was cross-linked to the enzyme CCA was still faithfully added to the tRNA's 3′ end [61].…”

Section: Primer Substrate Recognition and Processivitymentioning

confidence: 92%

“…Part of the ribonucleoprotein template pocket consists of a conserved β-turn structure which is structurally and functionally conserved in all CCAtrs [38]. A variant of this structure is also found in the bacterial PAPs where it is most likely involved in primer recognition and thus far is the only motif found to distinguish class II CCAtrs and PAPs [39].…”

Section: Structure Of Active Sites In Cca-adding Enzymesmentioning

confidence: 99%

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Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

Martín

Doublié

Keller

2008

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Poly(A) polymerases were identified almost fifty years ago as enzymes that add multiple AMP residues to the 3′ ends of primer RNAs without use of a template from ATP as cosubstrate and with release of pyrophosphate. Based on sequence homology of a signature motif in the catalytic domain, poly(A) polymerases were later found to belong to a superfamily of nucleotidyl transferases acting on a very diverse array of substrates. Enzymes belonging to the superfamily can add from single nucleotides of AMP, CMP or UMP to RNA, antibiotics and proteins but also homopolymers of many hundred residues to the 3′ ends of RNA molecules.The recently reported structures of several nucleotidyl transferases facilitate the study of the catalytic mechanisms of these very diverse enzymes. Numerous structures of CCA-adding enzymes have now revealed all steps in the formation of a CCA tail at the 3′ end of tRNAs. In addition, structures of poly(A) polymerases and uridylyl transferases are now available as binary and ternary complexes with incoming nucleotide and RNA primer. Some of these proteins undergo significant conformational changes after substrate binding. This is proposed to be an indication for an induced fit mechanism that drives substrate selection and leads to catalysis. Insights from recent structures of ternary complexes indicate an important role for the primer molecule in selecting the incoming nucleotide.

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Section: Primer Substrate Recognition and Processivitymentioning

confidence: 92%

Section: Structure Of Active Sites In Cca-adding Enzymesmentioning

confidence: 99%

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

Martín

Doublié

Keller

2008

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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“…Unlike poly(A) polymerases, CCA-adding enzymes not only add two different nucleotides in a defined order but terminate addition after three nucleotides (1,12,14). The cocrystal structures of two class II enzymes [the BstCCA enzyme in complex with CTP or ATP (14) and the A. aeolicus class II A-adding enzyme in complex with tRNA-NCC and a nonreactive ATP analog (21)] suggest that three residues in helix J (R194, M197, and E198) may constrain the growing 3Ј end of tRNA and/or help to thread it into the active site (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Other protocols (mutagenesis, protein expression, preparation of RNA substrates, and enzyme assays) have been described previously (12,22), and details are available upon request.…”

Section: Methodsmentioning

confidence: 99%

“…We therefore proposed that the growing 3Ј end of tRNA refolds progressively to reposition the new 3Ј hydroxyl identically relative to the single active site (10,12). This prediction was confirmed by cocrystal structures of the class I archaeal Archaeoglobus fulgidus CCAadding enzyme with oligonucleotide mimics of two different substrates (tRNA-NC ϩ CTP and tRNA-NCC ϩ ATP), as well as with mature tRNA-NCCA (where N is the discriminator base), which demonstrated that class I enzymes use a ribonucleoprotein template where the growing 3Ј terminus of tRNA works together with the protein to ''template'' addition of the next nucleotide (13).…”

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confidence: 99%

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Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases

Cho

Verlinde

Weiner

2007

Proc. Natl. Acad. Sci. U.S.A.

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CCA-adding enzymes build and repair the 3 -terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC-and A-adding enzymes into UU-and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCAadding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2 hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 Å away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3 end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.nucleotidyltransferase ͉ tRNA T he CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase (NTR)] builds and repairs tRNA by adding the nucleotide sequence CCA to the 3Ј terminus of immature or damaged tRNA (1). Although this unusual RNA polymerase has no nucleic acid template, it constructs the CCA sequence one nucleotide at a time using CTP and ATP as substrates (1). All CCA-adding enzymes and poly(A) polymerases belong to the NTR superfamily, which can be divided into two distinct classes according to sequence motifs in the catalytic domain (2-6). The class I and class II CCA-adding enzymes exhibit little if any homology outside the NTR domain (6). Class I NTRs include archaeal CCA-adding enzymes, eukaryotic poly(A) polymerases, and probably eukaryotic terminal uridylyltransferases (7, 8); class II NTRs include eukaryotic and eubacterial CCAadding enzymes as well as eubacterial poly(A) polymerases (6).We found previously that a single NTR motif adds all three nucleotides (9, 10), that tRNA does not rotate or translocate along the enzyme during addition of C75 and A76 (11), and that a single active subunit in these dimeric or tetrameric enzymes can carry out all three steps of CCA addition (9). We therefore proposed that the growing 3Ј end of tRNA refolds progressively to reposition the new 3Ј hydroxyl identically relative to the single active site (10,12). This prediction was confirmed by cocrystal structures of the class I archaeal...

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