Exchange of Regions between Bacterial Poly(A) Polymerase and the CCA-Adding Enzyme Generates Altered Specificities

Betat, Heike; Rammelt, Christiane; Martín, Georges; Mörl, Mario

doi:10.1016/j.molcel.2004.06.026

Cited by 48 publications

(76 citation statements)

References 28 publications

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“…10B). Thus, as previously observed (24), poly(A) polymerase is surprisingly nonspecific: The enzyme can add an A residue to immature tRNA-NCC in vivo (19), augmenting tRNA repair by the CCA-adding enzyme, but it can also add a poly(A) tail to mature tRNA in vitro (25) (SI Fig. 10B).…”

Section: Resultssupporting

confidence: 78%

“…The ability of this region to confer CCA-adding activity on the N terminus of poly(A) polymerase is most surprising and is consistent with the notion that scrunching, counting, and the nucleotide specificity switch in class II CCA-adding enzymes may be localized, intrinsic characteristics of the NTR active site; however, we hesitate to interpret the results of Betat et al (25) structurally because no structure has been determined for E. coli poly(A) polymerase; the helix M regions of the E. coli poly(A) polymerase and CCA-adding enzymes exhibit little if any homology; and helix M appears to be remote from the tRNA acceptor stem in the cocrystal structure of the A. aeolicus A-adding enzyme (21). (21), but homologous residues are labeled according to the BstCCA sequence (14), including M197, which is N197 in A. aeolicus.…”

Section: Resultssupporting

confidence: 78%

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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: 78%

Section: Resultssupporting

confidence: 78%

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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“…Furthermore, phylogenetic analysis indicates that collaborative CCA addition catalyzed by CC-and A-adding enzymes seems to be more widespread in bacteria than previously expected. In addition, the data not only support the idea of an ancestral CCA-adding enzyme but also indicate that nucleotidyltransferases are composed of individual modules that can be replaced by corresponding elements of related enzymes (5). Such combinations lead to proteins with new and often unexpected activities, ranging from partial to complete CCA addition.…”

Section: Discussion the Absence Of The Flexible Loop Region Is A Commsupporting

confidence: 57%

“…To address this question, the corresponding region of the CCA-adding enzyme of Bacillus subtilis (14) was introduced into the CCadding enzyme of B. halodurans. These two enzymes were chosen because they show a high sequence similarity (10), which increases the probability of catalytically active chimeric constructs, as it was successfully shown for other nucleotidyltransferase chimeras (5). For insertion, two highly conserved amino acid residues present in CC-as well as CCA-adding enzymes (E79, representing the postulated third catalytic carboxylate, and E89 in the B. halodurans enzyme) flanking the loop region were chosen as fusion positions.…”

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

confidence: 99%

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

Neuenfeldt

Just

Betat

et al. 2008

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

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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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“…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]. In addition, experiments with the E. coli CCAtr and PAP have shown that when the N-terminal catalytic and C-terminal RNA binding domains were exchanged between the two proteins, the PAP C-terminus allowed translocation of an RNA primer, thus resulting in a poly(CCA) tail added to tRNA when combined with the catalytic domain of CCAtr [62]. Conversely, when the C-terminal RNA binding domain of CCAtr was fused to the catalytic domain of bacterial PAP, the chimera acquired a CCA adding activity and added a single CCA.…”

Section: Primer Substrate Recognition and Processivitymentioning

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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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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Exchange of Regions between Bacterial Poly(A) Polymerase and the CCA-Adding Enzyme Generates Altered Specificities

Cited by 48 publications

References 28 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

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

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