Polynucleotide phosphorylase interacts with ribonuclease E through a betabetaalphabetabetaalpha domain.

In the present work we have used a double-hybrid assay in bacteria to identify a putative domain in E. coli PNPase required for in vivo interaction with RNase E. We used a 202 aa fragment of RNase E previously reported as the PNPase binding domain in this enzyme and a collection of 13 different fragments of 105 aa, spanning the entire sequence of 734 aa PNPase (GenBank Accession number NP_417633). Our results indicate that two clones of PNPase including residues 158-262 and residues 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration. Three-dimensional modeling of the E. coli PNPase based on the S. antibioticus protein structure indicates that the putative binding domain is located on the monomer surface, facing outward from the trimeric tertiary structure. Since a copy of the betabetaalphabetabetaalpha domain is also found in RNase PH, we investigated and found an interaction with RNase E in a pull-down assay. We suggest this interaction takes place through the similar betabetaalphabetabetaalpha domain present in the tertiary structure of this enzyme. Based on these results, we propose that RNase PH and RNase E could form functional assemblies in E. coli.

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