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. 2018 Jul 4;9:2613. doi: 10.1038/s41467-018-05053-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Ribosome profiling.a Schematic representation of translation initiation and assembly of the 80S ribosome. The 48S preinitiation complex is constituted of the 40S ribosomal subunit, eIF3 and the (eIF2)–GTP-tRNAP^MetP. Hydrolysis of GTP leads to release of eIF2. The L-shape of SLII is crucial for this process. Hence, the ISL mutant, misfolded WT and GMP-PNP, inhibiting GTPase activity, stall the reaction at this stage (bar-headed line). In contrast, miR-bound WT and Δ20 should activate eIF2-release, since SLII is effectively formed (green arrow). Subsequently, eIF3 is released and the 60S subunit is recruited to form the 80S complex. The following transition from initiation to elongation can be inhibited by CHX (bar-headed line). b Monitoring translation by luciferase assay in HeLa cell extracts, using Luc-SG in presence of miR^mut or miR^WT, DI mutants, GMP-PNP or CHX. c Analysis of HCV RNA distribution in ribosome profiles of HeLa cell extracts. Total lysates incubated with HCV in vitro transcripts were recorded; Relative abundance of HCV RNA from sucrose fractions was analyzed by qRT-PCR. Represented are percentages relative to the total amount in the gradient. The assay was performed with either no inhibitor, GMP-PNP or CHX in the reaction mix. The HCV RNA profile for the control reactions is shown in the top panel. Total RNA was analyzed by denaturing agarose gel electrophoresis (lower panel) to confirm the presence of 18S and 28S ribosomal RNA and distinguish precisely 40/48S and 80S ribosomes. Note that in fraction 4 and 9 the CHX data points are obscured by the GMP data. d Comparison of translation initiation efficiency of Luc-SG in presence of miR^WT. The control with miR^mut is shown as dashed line for comparison. e, f As in (d), comparing the WT reporter replicon and DI mutants. Mean values (±SD), n = 3, in technical duplicates. RLU relative light units. Significance was determined compared to the miR^mut control. The reference graph used to calculate is given in light gray. *P<0.05, **P<0.01, ***P<0.001. 40/48S and 80S containing fractions are highlighted by yellow or orange boxes, respectively. Note that results shown in panels (d–f) were obtained in absence of CHX