1. Exchange of Regions between Bacterial Poly(A) Polymerase and the CCA-Adding Enzyme Generates Altered Specificities 
Heike Betat, Christiane Rammelt, Georges Martin, Mario Mörl 
Molecular Cell 
Volume 15, Issue 3, Pages (August 2004)
DOI: /j.molcel
Copyright © 2004 Cell Press Terms and Conditions

2. Figure 1
Schematic Representation of the Parental Enzymes E. coli poly(A) Polymerase and CCA-Adding Enzyme
Poly(A) polymerase is shown in blue, and the CCA-adding enzyme is shown in green. Swap positions for chimeras CCA/PAP 1 and PAP/CCA 1 are indicated. The displayed N-terminal regions (residues 1–294 and 1–218, respectively) carry the active site signature of nucleotidyltransferases (presented in red boxes).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Enzyme Chimeras CCA/PAP 1 and CCA/PAP 2
(A) Proteins are represented as bars, with elements of the catalytic domain in red. The N-terminal part of the CCA-adding enzyme is indicated in green, and the C-terminal PAP region is indicated in blue. The putative RNA binding domain (RBD) of PAP is labeled in brown. While CCA/PAP 1 carries only parts of the RBD region, the complete domain is present in CCA/PAP 2.
(B) Activity test of CCA/PAP 1 and parental enzymes with tRNA lacking a CCA terminus (left panel) and with oligo(A)12 (right panel). Analysis by polyacrylamide gel electrophoresis revealed that CCA/PAP 1 adds multiple rounds of CCA exclusively on the tRNA substrate (products 1, 2, and 3), while parental enzymes show typical activities (CCA*: band representing CCA addition and CCCA incorporation, which is a known in vitro side product of the E. coli CCA-adding enzyme). CCA/PAP 2 showed identical activity (not shown).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Enzyme Chimeras PAP/CCA 1 and PAP/CCA 2
The color code in the upper panel is identical to that in Figure 2. In PAP/CCA 2, the fusion position is shifted toward the C terminus, thereby elongating the PAP region by 27 amino acids. The lower part shows gel electrophoresis of resulting reaction products.
(A) PAP/CCA 1 catalyzes the addition of CCA to a tRNA substrate and therefore acts as a true CCA-adding enzyme. CCA*: mixture of CCA and CCCA addition (a side product also observed in the parental CCA-adding enzyme of E. coli). The nature of the incorporated bases was analyzed by sequencing. Right panel: while PAP is active on oligo(A)12, PAP/CCA 1 does not accept this substrate.
(B) In contrast to PAP/CCA 1, PAP/CCA 2 exclusively incorporates ATP (verified by sequencing of reaction products) and accepts tRNA as well as oligo(A)12 as substrates. Thus, it represents typical PAP activity.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Summary of All Constructs and Chimeric Enzymes
The asterisks indicate the truncated PAP versions (amino acids 1–274 and 1–331) described by Raynal and Carpousis (Raynal and Carpousis, 1999). Enzymatic activity of individual enzyme versions is indicated on the right. The color code is the same as in Figure 2 (black representing the thioredoxin tag). While CCA N termini display exclusively CCA-adding activity, the activity of the corresponding part of PAP is context dependent and can be modified by the shift of the fusion position. Red dashed boxes indicate the 27 amino acid region that probably carries the anchor element in the CCA-adding enzyme and dictates the specificity of the catalytic core of poly(A) polymerase.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Cartoon to Illustrate the Concept of the Exchange of Modules between PAP and the CCA-Adding Enzyme, which Confers New Activities
Replaced regions of the E. coli enzymes were superimposed on the crystal structure of the B. stearothermophilus CCA-adding enzyme (Li et al., 2002). Swap positions (CCA/PAP 1: CCA1–218/PAP295–472; CCA/PAP 2: CCA1–198/PAP275–472; PAP/CCA 1: PAP1–294/CCA219–412; PAP/CCA 2: PAP1–321/CCA245–412) as well as deletion sites (CCA Δ1: CCA1–264, active [+]; CCA Δ2: CCA1–218, inactive [−]) are indicated. The C-terminal region of the CCA-adding enzyme (green) carries an unidentified anchor domain that restricts polymerization to the incorporation of three nucleotides. Replacement of the C terminus, including the anchor region, by the corresponding PAP region (blue) removes this restrictive domain and leads to a poly(CCA) polymerase activity (left). In the reciprocal exchange experiment, the resulting activity depends on the position of the fusion site (right): if helix M and the surrounding loop structures (red) are derived from the CCA-adding enzyme, the chimera acts as a true CCA-adding enzyme. If the helix M region is derived from PAP, the chimera displays PAP activity. Upper panel: secondary structure prediction of the original 27 amino acid regions of E. coli PAP and the CCA-adding enzyme. Both sequences are likely to fold into a helical domain similar to the structure of the B. stearothermophilus enzyme.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Nucleotide Binding Regions of Poly(A) Polymerase, in E. coli, and the CCA-Adding Enzyme, in E. coli and B. stearothermophilus
(A) Alignment of the active site and nucleotide binding region of investigated enzymes (EcoCCA and EcoPAP). As a reference, the sequence of the B. stearothermophilus CCA-adding enzyme (BstCCA) is shown. Gray boxes indicate conserved amino acids, and residues involved in nucleotide recognition are indicated in red. Poly(A) polymerase carries template amino acids identical to the CCA-adding enzymes. P: interaction with the triphosphate moiety, 2′OH: interaction with the 2′-hydroxyl group of the ribose; A, C: interaction with bases adenine and/or cytosine according to Li and coworkers (Li et al., 2002).
(B) Possible hydrogen bonds between template amino acids and base moieties (after Cho et al., 2003). Both CCA-adding enzymes and the poly(A) polymerase can form the same hydrogen bonds with the incoming ATP and CTP. 4-NH2 of CTP and 6-NH2 of ATP function thereby as H-donors and interact with a conserved aspartic acid side chain of the enzymes (D212, D131, D154). 3-N of CTP and 1-N of ATP act as H-acceptors for an invariant arginine residue (R215, R134, R157). In addition, the arginine residue can form another H-bond with the 2-O group of the cytidine. These highly conserved amino acid residues might explain the observed promiscuity of the catalytic domain of the poly(A) polymerase.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2004 Cell Press Terms and Conditions