Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element

Firth, Andrew E.; Wills, Norma M.; Gesteland, Raymond F.; Atkins, John F.

Plasmid DNAs (0.2 mg) were used as templates in 10 ml reactions of the rabbit reticulocyte lysate TNT Õ T7 Quick Coupled Transcription/Translation system (Promega). 35 S-methionine (Perkin Elmer) was included in the reactions and protein products were separated by SDS-PAGE. Dried gels were analyzed using a Typhoon PhosphorImager (GE Healthcare) and the amount of A nsP1 nsP2 nsP3 nsP4 C E3 E2 6K E1 stop codon readthrough −1 frameshift site n i e t o r p y l o p l a r u t c u r t s n i e t o r p y l o p l a r u t c u r t s n The plot depicts the probability that the degree of conservation within a 9-codon sliding window could be obtained under a null model of neutral evolution at synonymous sites. Note that the RT stop codon itself has been excluded from the conservation statistics. In order to map the conservation statistic onto the coordinates of a specific sequence in each alignment, all alignment columns with gaps in a chosen reference sequence were removed prior to calculation of conservation. The following reference sequences (GenBank accession numbers) were used: VEEV NC_001449, SINV NC_001547. radioactivity in each product was determined using the ImageQuant 5.2 program (Molecular Dynamics). After normalization for the number of methionine residues in termination and RT products (9 and 22, respectively), the RT efficiencies were calculated as [RT/ (RT+termination)].