Rapid Long Range Sliding of RNA Dependent RNA Polymerases on Viral Genome Templates Visualized by Photoactivatable Localization Microscopy

Abstract

While DNA binding proteins like the lac repressor have been observed to slide efficiently on the DNA template, the eukaryotic DNA dependent RNA polymerases were shown to bind their promoters directly from solution. Initiation of transcription remains less understood for negative sense RNA viruses which include major human and animal pathogens such as Ebola, measles, Rabies, VSV and Influenza virus. The viral genomic RNA is encapsidated in thousands of copies of nucleoprotein N (∼30kD), which can efficiently shield the full stretch of RNA genome and protect the RNA to be inaccessible even to the RNAses. To transcribe this genomic RNA, viruses package tens of copies of a specialized RNA dependent RNA polymerase which is composed of a catalytic subunit L (∼250kD) and an N-RNA binding protein p (∼30kD). While the full genome N-RNA can be longer than 4 microns, transcription can only initiate from the 3’ end. We have previously measured the binding affinity of the RdRPs to the N-RNA genome template and have shown that RdRPs have a dissociation constant of less that 20pM. Theoretical predictions of feasible transcription models all have pointed out to the essential requirement that the RdRPs slide on the N-RNA genome templates. Here we have created a recombinant VSV virus that has L protein fused with the Dendra2 fluorescent protein. We report tracking of single RdRPs along genome templates using photoactivatable localization microscopy. Our observations show rapid sliding of single RdRPs with diffusion coefficients as high as 106 nm2/sec and distances as long as 800 nanometers. These observations confirm theoretical studies predicting one-dimensional diffusion for RdRPs on VSV's genome templates, and also unmask a fundamental mechanism of redistribution of polymerases on the genome templates.