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. 2014 Apr 7;111(16):E1620–E1628. doi: 10.1073/pnas.1321660111

Fig. 7.

Fig. 7.

Effects of PMTC formation on TMV RNA translation. (A) Translation of purified PMTC in mdBYL. TMV RNA was translated with or without CHX in mdBYL, and the reaction mixtures were fractionated by SEC. The PMTC fraction and the control SEC fraction were each mixed with FLUC mRNA and were mixed with fresh mdBYL translation mixture containing [35S]-methionine and incubated (direct translation). On the other hand, an aliquot of each FLUC-supplemented fraction was deproteinized by phenol extraction, and purified RNA were translated in mdBYL in the presence of [35S]-methionine (translation after phenol extraction). Proteins were analyzed by SDS/PAGE and autography. The positions of the 126-kDa, 183-kDa, and FLUC proteins are shown on the right. (B) Time course of replication protein production from wild-type TMV and TMV-5′PVX RNAs in mdBYL. Translation reactions were performed with FLUC mRNA as an internal control but without exogenously added methionine. At time points indicated above the gels, [35S]-methionine was added, and, after a 5-min incubation, excessive (final, 1 mM) non–isotope-labeled methionine was added, and incubation was continued for a total of 90 min. Proteins were analyzed by SDS/PAGE and autoradiography. The positions of the 126-kDa and 183-kDa proteins are shown on the right. The graph shows the ratio of the FLUC-normalized 126-kDa protein band intensity for TMV (wt) to that for 5′PVX at each time point. (C) Effect of the Met-IR fragment binding on translation of reporter mRNA with viral 5′ UTRs. TMV-5′PVX-Met-IR-F or TMV-5′PVX-126-F RNA were translated in mdBYL for 30 min (for a negative control reaction, no mRNA was added). Then, Renilla luciferase mRNA that have 5′ UTR sequences from TMV RNA (TMV-RLUC) or that from TMV-5′PVX RNA (PVX-RLUC) were added to each mixture and incubated for an additional 60 min, and luciferase activity was measured. Boxes and error bars represent means and SEs of data obtained in three independent experiments. An asterisk indicates a significant difference by t test (P < 0.05).