Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Tomita, Kozo; Weiner, Alan M.

doi:10.1074/jbc.m207527200

Cited by 46 publications

(56 citation statements)

References 45 publications

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“…Similarly unexpected is the observation that k cat /K m for GTP addition by the G-adding enzyme is actually 5.3-fold improved relative to k cat /K m for ATP addition by the wild-type A-adding enzyme (SI Table 2). Taken together, these observations are more consistent with duplication of a CCA-adding gene to give CC-and A-adding activities than with evolution of a CCor A-adding enzyme into a true CCA-adding activity as originally proposed (18). The decreased catalytic efficiency of the A. aeolicus CC-and A-adding enzymes might then reflect the compromises required for the new nucleotide binding sites to exclude one NTP when the enzyme originally bound both, and for the new tRNA binding sites to exclude tRNAs of nearly comparable length when the site originally bound three different substrates (tRNA-N, tRNA-NC, and tRNA-NCC).…”

Section: Resultssupporting

confidence: 69%

“…In most eubacteria, CCA addition is carried out by a single CCA-adding enzyme, but in the deeply branching eubacteria Aquifex aeolicus and Deinococcus radiodurans (17,18), and possibly in the Gram-positive eubacterium Bacillus halodurans (19), related CC-and A-adding enzymes work sequentially to add CCA. All of these eubacterial CCA-, CC-, and A-adding enzymes, as well as eubacterial poly(A) polymerase and eukaryotic CCA-adding enzymes, belong to the same class II NTR superfamily (6,17,19,20); moreover, a sequence alignment of the nucleotide binding region (helices F and G) in class II enzymes (SI Fig.…”

Section: Resultsmentioning

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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Section: Resultssupporting

confidence: 69%

Section: Resultsmentioning

confidence: 99%

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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“…Surprisingly, these motifs for CCA addition are also present in a subset of bacterial tRNA nucleotidyltransferases that exert only partial activities (CC and A addition) and collaborate to synthesize a complete CCA end (8,9). This indicates that there must be other, not yet identified features responsible for the restricted nucleotide incorporation of these closely related enzymes.…”

mentioning

confidence: 98%

“…In Aquifex aeolicus, Deinococcus radiodurans, Bacillus halodurans, and Synechocystis sp., CCA synthesis is catalyzed by two nucleotidyltransferases (CC-adding and A-adding enzyme) in a stepwise addition of two C and one A residues (8)(9)(10). Most CC-adding enzymes show some deviation in the amino acid template EDxxR, where the first position (E) is replaced by R, A, or D, respectively.…”

Section: Cc-adding Enzymes Share the Deletion Of A Short Flexible Loopmentioning

confidence: 99%

“…1 A). Because former whole sequence alignments did not identify any features specific for CC-adding enzymes (6,9), the alignment was restricted to the region surrounding the loop position. Carrying motifs A and B as well as the conserved putative third carboxylate involved in catalysis (6,12), this specific region allows a refined and improved alignment of individual sequences.…”

Section: Cc-adding Enzymes Share the Deletion Of A Short Flexible Loopmentioning

confidence: 99%

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Evolution of tRNA nucleotidyltransferases: A small deletion generated CC-adding enzymes

Neuenfeldt

Just

Betat

et al. 2008

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

133

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CCA-adding enzymes are specialized polymerases that add a specific sequence (C-C-A) to tRNA 3 ends without requiring a nucleic acid template. In some organisms, CCA synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (CC and A addition). These enzymes carry all known motifs of the catalytic core found in CCA-adding enzymes. Therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete CCA terminus. Here, a region located outside of the conserved motifs was identified that is missing in CC-adding enzymes. When recombinantly introduced from a CCA-adding enzyme, the region restores full CCAadding activity in the resulting chimera. Correspondingly, deleting the region in a CCA-adding enzyme abolishes the A-incorporating activity, also leading to CC addition. The presence of the deletion was used to predict the CC-adding activity of putative bacterial tRNA nucleotidyltransferases. Indeed, two such enzymes were experimentally identified as CC-adding enzymes, indicating that the existence of the deletion is a hallmark for this activity. Furthermore, phylogenetic analysis of identified and putative CCadding enzymes indicates that this type of tRNA nucleotidyltransferases emerged several times during evolution. Obviously, these enzymes descend from CCA-adding enzymes, where the occurrence of the deletion led to the restricted activity of CC addition. A-adding enzymes, however, seem to represent a monophyletic group that might also be ancestral to CCA-adding enzymes. Yet, experimental data indicate that it is possible that A-adding activities also evolved from CCA-adding enzymes by the occurrence of individual point mutations.tRNA maturation ͉ CCA-adding enzyme ͉ flexible loop

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TransferRNASynthesis and Regulation

Toh

Hori

Tomita

et al. 2009

Encyclopedia of Life Sciences

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Transfer ribonucleic acid (tRNA) is primarily synthesized from tRNA gene through transcription by RNA polymerase and becomes the mature form via several steps: processing, splicing, CCA addition and posttranscriptional modification. Primary transcripts of tRNA genes contain 5′ and 3′ extra sequences, which are removed by a set of responsible nucleases, and introns in some cases, which are spliced out by a specific endonuclease . The resultant two fragments are joined by RNA ligase . The CCA sequences present at 3′‐termini of all mature tRNAs are not encoded in tRNA genes in some species and posttranscriptionally added by a CCA‐adding enzyme. All mature tRNA molecules contain modified nucleotides made by modification enzymes and which are considered to be involved in stabilization of tRNA structure, in decoding properties and in correct processing. The concentration of individual tRNA molecules is controlled to maintain cellular functions. Key concepts: tRNA is synthesized from tRNA gene by RNA polymerase and matured through processing, splicing, CCA addition and posttranscriptional modification. Synthesis of tRNA is regulated by promoter activity and specific factors (ppGpp and/or pppGpp in prokaryotes and Maf1 in eukaryotes) depending on the nutrient condition of the cells. The relative amounts of tRNA are regulated by several factors; the copy number of the tRNA gene, transcriptional activity aforementioned and tRNA degradation by a number of nucleases. Primary transcripts of tRNA genes contain 5′ and 3′ extra sequences, which are removed by a set of responsible nucleases. In some cases tRNA transcripts contain introns, which are spliced out by a specific endonuclease and the resultant two fragments are joined by RNA ligase. CCA‐adding enzyme regulates the amount of active tRNA by repairing CCA sequence at the C ‐terminal of tRNA. tRNA possesses a variety of modified nucleotides which are introduced by modification enzymes during or after the processing, splicing and transport steps. Several modifications in tRNA play important roles in the translation process, such as enhancement, expansion, restriction and/or alteration of codon–anticodon interactions, stabilization of tRNA structure, recognition by aminoacyl‐tRNA synthetase, etc.

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Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Cited by 46 publications

References 45 publications

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

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

Evolution of tRNA nucleotidyltransferases: A small deletion generated CC-adding enzymes

TransferRNASynthesis and Regulation

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