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. 2020 Aug 17;11:4134. doi: 10.1038/s41467-020-17974-z

Fig. 2. Stable ribosome stalling caused by the addition of the hCMV uORF2 stalling peptide downstream of the Rluc ORF.

Fig. 2

a Schematic representation of modified RLuc reporter mRNA constructs containing the hCMV uORF2 stalling peptide (SP + pA) or a non-stalling control sequence (CTR + pA). The red arrow denotes the radiolabeled toeprint primer and the orange dot the termination codon. The 200-nt long 3′UTR is followed by an 80 nt-long poly(A) tail depicted as A(80). b Toeprint analysis of ribosome occupancy at the TC of the hCMV uORF2 was performed after in vitro translation in HeLa cell lysates for 50 min at 33 °C of equimolar amounts of the two reporter mRNAs described in a. Sanger sequencing reactions were run in parallel (G, T, C, A) to locate the positions of the toeprints. The position of the TC and the toeprint band corresponding to the ribosomes at the TC (+18) are indicated. c Representative western blot analysis of an aliquot of the translation reactions analyzed in b. Equal amounts of the translation reactions were separated on a 12% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for tyrosine-tubulin (Tyr-Tub) and Rluc protein. The band corresponding to full-length Rluc protein is denoted by an arrow. The experiment was repeated three times. Source data are provided as a Source Data File.