Interactions of a Fluorescently‐Labeled Tobacco Mosaic Virus (TMV) RNA with Pokeweed Antiviral Protein (PAP)

Abstract

The 5′‐terminal 7‐methylguanosine (m7GpppN, where N is any nucleotide) cap structure of eukaryotic mRNAs is involved in RNA stabilization, transport, translation, and is recognized by many cellular proteins involved in pre‐mRNA splicing, RNA export, translation initiation and RNA turnover. Enzymatic production of capped RNA is an easy way to improve the stability and translational competence of RNA used for in vitro studies. Fluorescence spectroscopy is widely used to study these protein‐RNA interactions. The presence of a fluorescent cap derivative incorporated into the 5′ end of RNA provides an alternative method to study these cap‐binding interactions, and has several advantages: in general, fluorescent derivatives have a greater intensity than intrinsic protein (and/or nucleic acid) fluorescence; absorbance and emission maxima of the extrinsic fluorophore are far removed from native protein fluorescence, allowing interpretation of the interactions involving several proteins. Here, we describe a novel synthesis of a fluorescent cap analog, Anthranioyl‐GTP (Ant‐GTP), an in vitro cap labeling of a transcribed tobacco mosaic virus (TMV) RNA catalyzed by the recombinant vaccinia virus capping enzyme to produce functional Ant‐m7GpppG‐capped RNA. We further demonstrate efficient translation of this fluorescent RNA and study its interactions with pokeweed antiviral protein (PAP). PAP is a plant ribosome inactivating protein (RIP) produced by the common pokeweed plant. RIPs are RNA N‐glycosidases, and enzymatically remove purines from a universally conserved sarcin/ricin loop.