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. Author manuscript; available in PMC: 2019 Sep 6.
Published in final edited form as: Mol Cell. 2018 Aug 23;71(5):761–774.e5. doi: 10.1016/j.molcel.2018.07.028

Figure 4. Loss of Tma64 and Tma22 or Tma20 enhance reinitiation in cell extracts.

Figure 4.

(A) In vitro synthesized uORF-containing mRNAs (schematically shown in the middle) were translated in cell lysates prepared form three yeast strains in the absence (left panel) or in the presence (right panel) of the recombinant Tma20/Tma22 dimer. The luciferase activities were continuously measured and maximal rates of the product accumulation were calculated. In the left panel, the values were normalized to that obtained for the no_uORFluc construct in the same lysate and shown as a ratio to the rates in the WT lysate. On the right, luciferase activity was measured in the presence and absence of recombinant Tma protein. Luciferase levels in the presence of Tma factor were normalized to levels observed upon addition of buffer. Error bars, mean ±SD. (B) The same assay as in (A) was performed on bicistronic Rluc-Fluc mRNA constructs in the yeast lysates. A similar trend was observed, showing that these factors also function after long ORFs in this assay. For all panels, 3 replicates each containing 2 or more mRNA preparations were used for each experiment.