Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2014 Dec 17;43(1):446–460. doi: 10.1093/nar/gku1292

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

Figure 6. — Functional characterization of the effects of mutations in the MNV RdRp for RNA synthesis, binding to the Mps and their thermodenaturation profiles. (A) Purified recombinant proteins used in the analysis. The image is a SDS-PAGE stained with Coomassie blue. The molecular weight marker (M) is from Pierce. R411AR416A denotes that the recombinant RdRp contained alanine substitutions at both R411 and R416. (B) RNAs synthesized by the WT and mutant MNV RdRps. The concentration of WT or mutated RdRps is 250 nM and the Mps is at 50 nM in each reaction. The products of primer extension (PE) and de novo initiation (DN) are shown. The DN products are of 11- and 10-nt in lengths. The amount of RNAs normalized to that of the WT were quantified and shown below the gel image. (C) RNA synthesized by the WT and mutant MNV RdRps. The concentration of the WT or mutant RdRps is 1 μM and the Mps is at 50 nM in each reaction. (D) Increasing template Mps concentration was able to restore the amount of de novo-initiated RNA synthesis by R411A RdRp (upper graph). Interestingly, the relative amounts of primer-extension products were not corrected for the R411A mutant. (E) Binding isotherms for Mps by the WT, R411 and R416 RdRps. The dissociation constants derived from the binding isotherms are 0.5, 1.4 and 1.7 μM for the WT MNV, the R411A and R416A mutants. The data were derived from fluorescent polarization assays performed with a 6-FAM-labeled Mps and recombinant RdRps in the absence and presence of Mps. (F) The thermodenaturation profile of WT, R411A, R416A and R411AR416A RdRps. The upper panel shows a comparison of the thermodenaturation profiles of the WT MNV RdRp with R411A, R416A, R411AR416A mutants. The R411A RdRp has a T_mapp 2°C lower than that of the WT RdRp; R416A mutatns has a T_mapp 1°C higher than that of the WT RdRp; R411AR416A has the same T_mapp as that of WT RdRp. (G) The thermodenaturation profiles of WT RdRp without and with Mps. (H) Thermodenaturation profiles of R411A RdRp without and with Mps. The assay was performed with a 1:2 molar ratio of RdRp to RNA.