Polynucleotide phosphorylase binds to ssRNA with same affinity as to ssDNA

• Published in: Biochimie Vol. 84; no. 4; pp. 321 - 328
• Main Authors: Bermúdez-Cruz, Rosa Ma, García-Mena, Jaime, Montañez, Cecilia
• Format: Journal Article
• Language: English
• Published: France Elsevier Masson SAS 01.04.2002
• Subjects: 5' Untranslated Regions - genetics; Autoregulation; Base Sequence; Binding Sites; Dissociation constant; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Electrophoretic Mobility Shift Assay; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Kinetics; Linear Models; Molecular Sequence Data; Plasmids; Polynucleotide phosphorylase; Polyribonucleotide Nucleotidyltransferase - metabolism; Protein Binding; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA/DNA binding activity; Autoregulation; cpm; 152LR(+)/152LR(–) RNA; ss –447/–419(+)/ss –447/–419(–) DNA; ssRNA/DNA; Dissociation constant; 213LR DNA; K d; PNPase; RNA/DNA binding activity; RNase III/E/II; Pi; Polynucleotide phosphorylase; dsDNA
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/S0300-9084(02)01385-8

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is a multifunctional protein, with a 3′–5′ processive exoribonuclease, a Pi exchange, an RNA polymerase and an autoregulatory activity. The interaction between this enzyme and the mRNA target is crucial for its activities. In the present study, we characterized the interaction of PNPase with its mRNA regulatory region and ssRNA, as well as with ssDNA and dsDNA by determining K d. Our results indicate that PNPase has high affinity for its mRNA, ssRNA and for ssDNA ( K d∼10–20 nM). However, this enzyme exhibits a lower affinity for dsDNA ( K d∼200–1400 nM). Possible implications of these results on the molecular mechanisms by which PNPase is regulated and degrades mRNA are discussed.