Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

Abstract

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.HCV IRES | translation initiation | human ribosomes | single-molecule FRET P rotein synthesis is a central process in health and disease (1, 2). The basic steps in translation have been mapped by genetic, biochemical, structural, and mechanistic studies. However, how translation is regulated and subverted, for example, during viral infection, remains poorly understood, especially in eukaryotes. All viruses compete for the cellular translation machinery to synthesize viral proteins required for virus proliferation. To that end, many viruses contain a structured internal ribosome entry site (IRES) in the 5′ untranslated region of their genome, which allows them to bypass the requirement for certain translation initiation factors. How IRESs achieve this goal remains unclear.Recently, it has been shown that structurally and evolutionarily very diverse IRESs, such as hepatitis C virus (HCV) IRES, cricket paralysis virus (CrPV) IRES (3), and others (4), require ribosomal protein eS25 (RPS25) for efficient translation initiation, indicating that RPS25/IRES interactions could be a universal feature of IRES-mediated translation. RPS25 is located on the back of the head of the 40S ribosomal subunit, distal to the mRNA entry channel but proximal to different IRES RNAs as shown by cryo-EM structures of 80S:CrPV IRES and 80S:HCV IRES complexes. RPS25 is not essential for cap-dependent translation, suggesting that IRES/RPS25 interactions are required to bypass the requirement for the full set of initiation factors (3).Translation initiation is a multistep process, with kinetics and dynamic interrogation refractory to conventional biochemical and biophysical methods. Single-molecule approaches provide insight into compositional and conformational dynamics of these asynchronous processes by following individual molecular events in real time. In bacteria, single-molecule methods allow direct obser...

Dynamics of IRES-mediated translation

“…All cryo-EM structural models to date have suggested that the 40S : IRES complex exists in a single conformation. By contrast, the smFRET experiments observed domain II sampling at least two conformations with respect to the 40S subunit head [96] (figure 5d). These motions may reflect functionally relevant conformational changes, such as rotation of the 40S subunit head or domain II occupancy of the E site, similar to conformations adopted in 80S : IRES complexes.…”

“…These features make IRESmediated translation extremely well suited for singlemolecule studies, which can reveal the compositional and conformational dynamics of macromolecule assemblies [69]. Single-molecule FRET can measure the time evolution of distance changes during translation, such as ribosomal rotation during bacterial elongation [22] and 40S head motions during initiation [96]. However, FRET provides only a single distance constraint on a biochemical system requiring synergistic integration of detailed static structural data from cryo-EM [137,138].…”

“…Dynamic tools for investigation of human translation in vitro will continue to improve [96,139], and will be integrated with in vivo genome wide profiling studies [140] of eukaryotic translation. The study of IRES-mediated translation through structure and dynamics will be a springboard to extending these approaches to the general mechanisms of translation initiation and its regulation.…”

“…As eS25 is non-essential for cells cultured in vitro, the fluorescently labelling of human 40S ribosomes has now been accomplished, via the genetic complementation of a SNAPtag RPS25 gene fusion to a CRISPR/Cas9-mediate knockout cell line of RPS25 [96]. Investigation of the fluorescently labelled 40S subunit as a complex and the HCV IRES, labelled upstream of domain II, enabled single-molecule FRET (smFRET) analysis of the binary complex (figure 5d).…”

“…500 s), indicating that binary-complex formation is irreversible on the timescale of initiation [96]. The binary complex was first visualized at low resolution (20 Å ) by cryo-EM, showing that the IRES binds the rabbit 40S subunit in a single conformation mediated largely by interactions of domain III at the solvent side [97].…”