Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases.
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 CCA-adding 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 A 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.
Cho
Hyundae D
HD
Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA 98195-7350, USA.
Verlinde
Christophe L M J
CL
Weiner
Alan M
AM
D001665:Binding Sites; D006860:Hydrogen Bonding; D011061:Poly A / Q000378:metabolism*; D011066:Poly C / Q000378:metabolism*; D011068:Poly G / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000502:physiology*
D011061:Poly A; D011068:Poly G; C023795:poly(U-G); D011072:Poly U; C023794:poly(A-C); D011066:Poly C; C440000:A adding enzyme; C439999:CC adding enzyme; D012316:RNA Nucleotidyltransferases; C022413:poly G polymerase; C023198:tRNA nucleotidyltransferase; D011062:Polynucleotide Adenylyltransferase