Characterization of the RNA binding energetics of the Candida albicans poly(A) polymerase.
The 3' ends of eukaryotic mRNAs are characterized by the presence of a poly(A) tail, which plays a critical role in stability, transport, and translation of the mRNAs. In the present study, we report the expression, purification and enzymatic characterization of the poly(A) polymerase of Candida albicans, an important human pathogen. As a first step toward elucidating the nature of the interaction between RNA and the enzyme, fluorescence spectroscopy assays were also performed to monitor the binding of RNA to the protein. Our assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of the enzyme is eight nucleotides. Moreover, both the kinetics of real-time RNA binding and the contribution of electrostatic interactions to the overall binding energy were investigated. Finally, we also correlated the effect of RNA binding on protein structure, using both circular dichroism and guanidium hydrochloride-induced denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein. In addition to the determination of the energetics of RNA binding, our study provides a better understanding of the molecular basis of RNA binding by poly(A) polymerases.
Bougie
Isabelle
I
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Bisaillon
Martin
M
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D026723:Polyadenylation; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D012331:RNA, Fungal / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics
D012331:RNA, Fungal; D011062:Polynucleotide Adenylyltransferase