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Kinetic Pathway of 40S Ribosomal Subunit Recruitment to Hepatitis C

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Kinetic Pathway of 40S Ribosomal Subunit Recruitment to Hepatitis C Kinetic pathway of 40S ribosomal subunit recruitment INAUGURAL ARTICLE to hepatitis C virus internal ribosome entry site Gabriele Fuchsa,1, Alexey N. Petrovb,1, Caleb D. Marceaua, Lauren M. Popova, Jin Chenb,c, Seán E. O’Learyb, Richard Wangb, Jan E. Carettea, Peter Sarnowa, and Joseph D. Puglisib,2 aDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124; bDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and cDepartment of Applied Physics, Stanford University, Stanford, CA 94305-4090 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014. Contributed by Joseph D. Puglisi, November 10, 2014 (sent for review August 26, 2014; reviewed by Peter Cornish, Christopher S. Fraser, and Jeffrey S. Kieft) Translation initiation can occur by multiple pathways. To delineate fluorescently labeled components of translation initiation has pre- these pathways by single-molecule methods, fluorescently labeled vented application of the single-molecule methods to study the ribosomal subunits are required. Here, we labeled human 40S mechanism of translation initiation in humans. ribosomal subunits with a fluorescent SNAP-tag at ribosomal Here, we demonstrate an approach to create fluorescently la- protein eS25 (RPS25). The resulting ribosomal subunits could be beled 40S ribosomal subunits from human cells. Using these sub- specifically labeled in living cells and in vitro. Using single-mole- units, we measure the kinetics and conformational pathway of 40S cule Förster resonance energy transfer (FRET) between RPS25 and subunit recruitment to the HCV IRES. First, we created RPS25 domain II of the hepatitis C virus (HCV) internal ribosome entry site KO cell lines using the clustered regularly interspaced short pal- (IRES), we measured the rates of 40S subunit arrival to the HCV indromic repeat (CRISPR)-Cas system. Next, we expressed RPS25 IRES. Our data support a single-step model of HCV IRES recruit- as a C-terminal fusion with mutant O6-alkylguanine DNA alkyl- ment to 40S subunits, irreversible on the initiation time scale. We transferase (SNAP) as the sole source of the protein. We estab- furthermore demonstrated that after binding, the 40S:HCV IRES lished biochemically that RPS25-SNAP is efficiently incorporated complex is conformationally dynamic, undergoing slow large-scale into functional 40S subunits, and rescues the defect in HCV rearrangements. Addition of translation extracts suppresses these IRES-mediated translation caused by RPS25 deletion. Using BIOPHYSICS AND fluctuations, funneling the complex into a single conformation on single-molecule fluorescence, we showed that Förster resonance COMPUTATIONAL BIOLOGY the 80S assembly pathway. These findings show that 40S:HCV IRES energy transfer (FRET) between RPS25 and the base of domain complex formation is accompanied by dynamic conformational II of the HCV IRES is dynamic. Conformational dynamics were rearrangements that may be modulated by initiation factors. altered by the presence of translational extract, thus indicating that conformational flexibility of RPS25 and domain II positions HCV IRES | translation initiation | human ribosomes | single-molecule FRET could be responsible for guiding downstream steps of translation initiation on HCV IRES. Finally, by measuring the kinetics of the rotein synthesis is a central process in health and disease (1, 40S:HCV IRES interactions, we demonstrate that the rate of 40S P2). The basic steps in translation have been mapped by ge- subunit recruitment to the HCV IRES is not limited by the con- netic, biochemical, structural, and mechanistic studies. However, formational flexibility of the free HCV IRES, and that upon 40S how translation is regulated and subverted, for example, during binding, the 40S:HCV IRES complexes are stable on the time viral infection, remains poorly understood, especially in eukar- yotes. All viruses compete for the cellular translation machinery Significance to synthesize viral proteins required for virus proliferation. To that end, many viruses contain a structured internal ribosome entry Protein biosynthesis is most tightly controlled during trans- site (IRES) in the 5′ untranslated region of their genome, which lation initiation that involves numerous initiation factors and allows them to bypass the requirement for certain translation ini- regulatory proteins. This complexity confounds conventional tiation factors. How IRESs achieve this goal remains unclear. biochemical methods. Single-molecule approaches are ideally Recently, it has been shown that structurally and evolution- suited to address such questions. However, their application is arily very diverse IRESs, such as hepatitis C virus (HCV) IRES, hindered by the lack of fluorescently labeled components of cricket paralysis virus (CrPV) IRES (3), and others (4), require the eukaryotic translation machinery. Here, we demonstrate an ribosomal protein eS25 (RPS25) for efficient translation initia- approach to label human 40S ribosomal subunits. As an ex- tion, indicating that RPS25/IRES interactions could be a univer- tension of this approach, we used single-molecule fluorescence sal feature of IRES-mediated translation. RPS25 is located on the to demonstrate that 40S ribosomal subunits are recruited to back of the head of the 40S ribosomal subunit, distal to the the hepatitis C virus mRNA in a single-step process, and that mRNA entry channel but proximal to different IRES RNAs as components of a translational extract regulate the conforma- shown by cryo-EM structures of 80S:CrPV IRES and 80S:HCV tion of this complex. IRES complexes. RPS25 is not essential for cap-dependent translation, suggesting that IRES/RPS25 interactions are re- Author contributions: G.F., A.N.P., J.E.C., P.S., and J.D.P. designed research; G.F., A.N.P., L.M.P., and R.W. performed research; C.D.M. contributed new reagents/analytic tools; G.F. quired to bypass the requirement for the full set of initiation and A.N.P. analyzed data; and G.F., A.N.P., C.D.M., L.M.P., J.C., S.E.O., J.E.C., P.S., and J.D.P. factors (3). wrote the paper. Translation initiation is a multistep process, with kinetics and Reviewers: P.C., University of Missouri; C.S.F., University of California, Davis; and J.S.K., dynamic interrogation refractory to conventional biochemical and University of Colorado Denver School of Medicine. biophysical methods. Single-molecule approaches provide insight The authors declare no conflict of interest. into compositional and conformational dynamics of these asyn- Freely available online through the PNAS open access option. chronous processes by following individual molecular events in 1G.F. and A.N.P. contributed equally to this work. real time. In bacteria, single-molecule methods allow direct ob- 2To whom correspondence should be addressed. Email: [email protected]. servation of the multiple initiation pathways and conformational This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rearrangements that guide translation initiation (5, 6). The lack of 1073/pnas.1421328111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1421328111 PNAS | January 13, 2015 | vol. 112 | no. 2 | 319–325 Downloaded by guest on October 1, 2021 scale of initiation. This study demonstrates the power of real-time into the KO cells, creating KO + RPS25-SNAP cells. Expression observations of conformational and compositional dynamics of the 43-kDa RPS25-SNAP fusion protein was confirmed by during human translation initiation. immunoblotting with anti-RPS25 antibody. RPS25 abundances in the add-back cell lines corresponded to about 110% and 75% Results expression in Hap1 cells (Fig. 1B). To apply single-molecule approaches, fluorescently labeled ri- To detect RPS25-SNAP directly in living cells and in vitro, bosomal particles, translation factors, and tRNAs are required. RPS25 KO and KO + RPS25-SNAP cells or cell lysates were Bacterial ribosomes have been fluorescently labeled by either treated with SNAP-fluorescein and SNAP-649 dyes. Total cell protein- or nucleic acid-based attachment of dyes at specific lysates were separated by SDS/PAGE, and gels were scanned for locations. Mutants of ribosomal proteins containing single-Cys fluorescence. No fluorescence was detected in the RPS25 KO or residues can be reacted with maleimide-conjugated dyes. La- in the KO RPS25-SNAP add-back cell lines in the absence of dye. beled and purified proteins are then incorporated into pre- However, RPS25-SNAP was detected in the presence of either assembled ribosomes lacking these specific proteins (7, 8). A dye (Fig. 1C)inKO+ RPS25-SNAP lysates, indicating that second strategy exploits the introduction of metastable hairpins RPS25-SNAP labeling is specific. into surface-exposed loops of rRNA (9). Fluorescently labeled DNA oligonucleotides are then annealed to these hairpins, Intracellular Localization of RPS25-SNAP. Expression of a tagged resulting in site-specific labeled ribosomal particles. This ap- protein can cause it to misfold and lead to artifacts in its sub- proach has been used successfully to generate fluorescently la- cellular localization. Using confocal microscopy, we examined beled 40S ribosomal subunits from Saccharomyces cerevisiae (10). the cellular localization of RPS25-SNAP. RPS25-SNAP was vi- However, these approaches are difficult to implement in mam- sualized by adding a SNAP-fluorescein dye
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