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. 2018 Nov 5;47(1):299–309. doi: 10.1093/nar/gky1058

Figure 1.

Figure 1.

RNA synthesis and capping are controlled by distinct residues in a loop structure of the VSV L PRNTase domain. (A) A 3D structural model of the RdRp (cyan) and PRNTase (green) domains of VSV L is shown (top) in cartoon form with a close-up view of the large loop (orange) from the PRNTase domain is shown below. Top: Within the red box, each active site is labeled and active site residues are shown in stick model. B and D labels note PRNTase motifs B and D containing active site residues T1157 and H1227/R1228, respectively. Bottom: The loop with flanking residues is illustrated with surrounding PRNTase (T1157, H1227 and R1228) and RdRp (D605, D714) active site residues and residues on the loop shown in stick model. Models were generated from PDB id: 5A22. (B) A sequence logo [WebLogo (39)] for the loop flanking PRNTase motif B in rhabdoviral L proteins is shown with the corresponding sequences of VSV and RABV (numbers: amino acid positions). Ψ, Φ, π, [−], [+] and x indicate aliphatic, hydrophobic, small, negatively and positively charged, and any amino acids, respectively. (C, D) In vitro transcription was performed with VSV L [wild-type (WT) or mutant], P, and N-RNA. 32P-Labeled LeRNA and mRNAs (deadenylated) were analyzed by 20% (C) and 5% (D) urea-PAGE, respectively, followed by autoradiography. The positions of the origin (ori.) and previously identified transcripts are denoted. (E) In vitro capping was performed with pppAACAG, [α-32P]GDP and L (WT or mutant). Capped RNA products were analyzed by 20% urea-PAGE followed by autoradiography. (F) L (WT or mutant) was incubated with 32P-labeled pppAACAG. The resulting L-pRNA intermediate was analyzed by 7.5% SDS-PAGE. The graphs show relative activities for respective reactions, where radioactivities of respective products without (lanes 1 and 8) and with (lanes 2 and 9) of WT L were set to 0 and 100%, respectively. The dot-plots, columns, and error bars represent the individual normalized values, means, and standard deviations, respectively, from three independent experiments (n = 3).