Table 1.

Quantitative Analysis of in Vitro Translation Products

RNARelative Amount of uORF PeptideaRelative Amount of LuciferaseaRelative Amount of Luciferase from ReinitiationbReinitiation Frequency in Vitro (%)c
LUCWT1331004030
LUCcontxt212595124
LUCm123d227
LUCint1021301034333
LUCnew112816910985
LUCnew213218312393
LUCnew2contxt20317416682
  • a Values have been adjusted for the number of labeled methionine residues in each product, with the relative molar amount of luciferase in LUCWT set as 100. Each value represents the average of at least four independent SDS-PAGE analyses.

  • b Relative amount of luciferase from reinitiation is calculated by subtracting the luciferase from leaky scanning from the relative amount of luciferase for each construct. The fraction of luciferase from leaky scanning is 60% of 100 (LUCWT) for wild-type uORF-containing RNA and 8% of 100 (LUCWT) for constructs containing the uORF with the context improvement (see Figure 4).

  • c Reinitiation frequency in vitro is calculated by dividing the relative amount of luciferase from reinitiation by the relative amount of uORF peptide for each construct.

  • d (−), does not apply to this RNA.