The CCA-adding enzyme (tRNA nucleotidyltransferase) builds and repairs the 3 end of tRNA. A single active site adds both CTP and ATP, but the enzyme has no nucleic acid template, and tRNA does not translocate or rotate during C75 and A76 addition. We modeled the structure of the class I archaeal Sulfolobus shibatae CCA-adding enzyme on eukaryotic poly(A) polymerase and mutated residues in the vicinity of the active site. We found mutations that specifically affected C74, C75, or A76 addition, as well as mutations that progressively impaired addition of CCA. Many of these mutations clustered in an evolutionarily versatile ␤-turn located between strands 3 and 4 of the nucleotidyltransferase domain. Our mutational analysis confirms and extends recent crystallographic studies of the highly homologous Archaeoglobus fulgidus enzyme. We suggest that the unusual phenotypes of the ␤-turn mutants reflect the consecutive conformations assumed by the ␤-turn as it presents the discriminator base N73, then C74, and finally C75 to the active site without translocation or rotation of the tRNA acceptor stem. We also suggest that ␤-turn mutants can affect nucleotide selection because the growing 3 end of tRNA must be properly positioned to serve as part of the ribonucleoprotein template that selects the incoming nucleotide.The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) is the only RNA polymerase that can build or rebuild a specific nucleic acid sequence without using a nucleic acid template (1, 2). CCA-adding enzymes belong to the nucleotidyltransferase superfamily (3, 4), which can be subdivided into class I and class II according to highly conserved features of the nucleotidyltransferase motif (5) that positions two metal ions for the nearly universal phosphoester bond transfer mechanism (6, 7). Archaeal CCA-adding enzymes belong to class I, and eukaryotic and eubacterial CCA-adding enzymes belong to class II (5). Most surprisingly, the class I and class II CCAadding enzymes exhibit strong core homology within each class (about 40 kDa in class I and 25 kDa in class II) but little obvious homology between the two classes outside the immediate vicinity of the nucleotidyltransferase motif. A further complication is that CCA-adding activity is the joint responsibility of related class II CC-and A-adding enzymes in certain deeply rooted eubacteria (8, 9). The CCA-adding enzyme is a nonessential repair enzyme in those eubacteria whose tRNA genes all encode CCA (10, 11), but is an essential enzyme in eukaryotes (12) and presumably also in those Archaea and eubacteria whose tRNA genes do not all encode CCA.How does the CCA-adding enzyme build CCA without using a nucleic acid template? We found previously that tRNA does not translocate along the CCA-adding enzyme during addition of C75 and A76 (addition of C74 was not examined) (13) and that a single nucleotidyltransferase motif is responsible for adding all three nucleotides (14). (The 3Ј-terminal tRNA sequence NCCA is designated positions 73, 74, 75, and 76 in the univer...

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...

We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N 6 -methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2-O-methyladenosine, 2-deoxy-2-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.

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.

Two-dimensional paper networks (2DPNs) have enabled the use of paper-based platforms to perform multistep immunoassays for detection of pathogenic diseases at the point-of-care. To date, however, detection has required the user to provide multiple signal enhancement solutions and been limited to protein targets. We solve these challenges by using mathematical equations to guide the device design of a novel 2DPN, which leverages multiple fluidic inputs to apply fully dried solutions of hydrogen peroxide, diaminobenzidine, and horseradish peroxidase signal enhancement reagents to enhance the limit-ofdetection of numerous nucleic acid products. Upon rehydration in our unique 2DPN design, the dried signal enhancement solution reduces the limit-of-detection (LOD) of the device to 5 × 10 11 nucleic acid copies/mL without increasing false positive detection. Our easy-to-use device retains activity after 28 days of dry storage and produces reliable signal enhancement 40 min after sample application. The fully integrated device demonstrated versatility in its ability to detect double-stranded and single-stranded DNA samples, as well as peptide nucleic acids.