Fernández-Ramírez, F, Bermúdez-Cruz, RM and Montañez, C (2010) Nucleic acid and protein factors involved in Escherichia coli polynucleotide phosphorylase function on RNA. Biochimie 92:445-54
It has been reported that polynucleotide phosphorylase (PNPase) binds to RNA via KH and S1 domains, and at least two main complexes (I and II) have been observed in RNA-binding assays. Here we describe PNPase binding to RNA, the factors involved in this activity and the nature of the interactions observed in vitro. Our results show that RNA length and composition affect PNPase binding, and that PNPase interacts primarily with the 3' end of RNA, forming the complex I-RNA, which contains trimeric units of PNPase. When the 5' end of RNA is blocked by a hybridizing oligonucleotide, the formation of complex II-RNA is inhibited. In addition, PNPase was found to form high molecular weight (>440 kDa) aggregates in vitro in the absence of RNA, which may correspond to the hexameric form of the enzyme. We confirmed that PNPase in vitro RNA binding, degradation and polyadenylation activities depend on the integrity of KH and S1 domains. These results can explain the defective in vivo autoregulation of PNPase71, a KH point substitution mutant. As previously reported, optimal growth of a cold-sensitive strain at 18 degrees C requires a fully active PNPase, however, we show that overexpression of a novel PNPaseDeltaS1 partially compensated the growth impairment of this strain, while PNPase71 showed a minor compensation effect. Finally, we propose a mechanism of PNPase interactions and discuss their implications in PNPase function.
Base Sequence; Blotting, Northern; Blotting, Western; DNA Primers; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Molecular Weight; Polyribonucleotide Nucleotidyltransferase/metabolism; RNA, Bacterial/genetics; RNA, Bacterial/metabolism
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