The conserved A₆₂₀₅ is important for IRES-mediated translation.

<p>(A) Sequence alignment of the variable loop region (VLR) of members of the type I IGR IRESs. Nucleotides involved in basepairing of the helical stem are bolded and those comprising pseudoknot I are highlighted in grey. The nucleotides constituting the VLR are denoted, with conserved positions highlighted in red. The assignments are made based on the secondary structure predictions of the IGR IRESs. The translational start site is indicated. (B) The schematic of the PKI domain is shown with the nucleotides constituting the VLR highlighted in grey. Four conserved nucleotides within the VLR, A₆₂₀₅, A₆₂₀₇, A₆₂₀₈ and U₆₂₁₁ are denoted by boxes and were mutated to each of the alternate Watson-Crick bases. (C) Translational activities of VLR point mutants. Uncapped dicistronic reporter RNAs containing the wild-type or mutant IGR IRES in the intercistronic space were incubated in RRL in the presence of [³⁵S]-methionine. Shown are the average values ± S.D. for the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES from at least three independent experiments. (D) Toeprinting/primer extension assay of VLR point mutants. Dicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in RRL under treatment with edeine [E] or cycloheximide [C] as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051477#s2" target="_blank">Materials and Methods</a> section. Primer extension analysis was performed using PrEJ69 in the presence of α- [³²P]-dATP. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The sequencing reactions for the wild-type IRES are shown on the <i>left</i>, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the <i>right</i>. Shown is a representative gel from at least two independent experiments.