Specific interaction of zinc finger protein Com with RNA and the crystal structure of a self-complementary RNA duplex recognized by Com.

Martyna Nowacka,Grzegorz Lach,Agata Bernat,Alexander P Gultiaev,Humberto Fernandes,Janusz M Bujnicki,Agnieszka Kiliszek

doi:10.1371/journal.pone.0214481

Abstract

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com–RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.

Highlights

RNA-binding proteins play important roles at every stage of RNA life cycle: transcription, splicing, editing, export, degradation and regulation of translation [1]
After five cycles of Systematic Evolution of Ligands by Exponential enrichment (SELEX), we found two very similar RNA motifs, both with the core sequence 50-GAG(N)2CC-30, where N refers to any nucleotide (Fig 2A)
Com is a small zinc finger protein that regulates the translation of bacteriophage Mu Mom RNA

Summary

Introduction

RNA-binding proteins play important roles at every stage of RNA life cycle: transcription, splicing, editing, export, degradation and regulation of translation [1]. Specific interaction of Com with RNA program 01/KNOW2/2014 to AK) and by the National Science Center The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion