Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

Shenvi, Christina; Dong, Ken C.; Friedman, Eric; Hanson, Jeffrey; Cate, J.H.D.

doi:10.1261/rna.2192805

Cited by 50 publications

(41 citation statements)

References 67 publications

(82 reference statements)

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“…3A). Translation initiation in translation-competent extracts is usually inhibited at magnesium concentrations >2.5 mM (Shenvi et al 2005). Given the results reported here, however, high magnesium concentrations eems to primarily inhibit factor-dependent translation initiation.…”

Section: Hcv Ires Facilitates Translation Initiation In the Absence Osupporting

confidence: 46%

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Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site

Lancaster¹,

Jan²,

Sarnow³

2006

RNA

103

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The hepatitis Cviral mRNA initiates translation using an internal ribosome entry site (IRES) located in the 5 9 noncoding region of the viral genome. At physiological magnesium ion concentrations, the HCV IRES forms abinary complex with the 40S ribosomal subunit, recruits initiation factor eIF3 and the ternary eIF2/GTP/Met-tRNA i Met complex, and joins 60S subunits to assemble translation-competent 80S ribosomes. Here we show that in the presence of 5mMMgCl 2 ,the HCV IRES can initiate translation by an alternative mechanism that does not require known initiation factors. Specifically, the HCV IRES was shown to initiate translation in ar econstituted system consisting only of purified 40S and 60S subunits, elongation factors, and aminoacylated tRNAs at high magnesium concentration. Analyses of assembled complexes supported am echanism by which preformed 80S ribosomes can assemble directly on the HCV IRES at high cation concentrations. This mechanism is reminiscent of that employed by the divergent IRES elements in the Dicistroviridae,e xemplified by the cricket paralysis virus, which mediates initiation of protein synthesis without initiator tRNA.

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Section: Hcv Ires Facilitates Translation Initiation In the Absence Osupporting

confidence: 46%

“…Our results indicate that the HCVI RES has ag reater affinity for the 80S ribosomes at 5m MM g 2+ than at 2.5 mM Mg 2+ .I ti sp ossible that the ribosome has an altered conformation at higher magnesium concentration (Shenvi et al 2005), which can more readily form ac omplexw ith the HCVI RES.…”

Section: Discussionmentioning

confidence: 68%

Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site

Lancaster¹,

Jan²,

Sarnow³

2006

RNA

103

View full text Add to dashboard Cite

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“…Because the total cytosolic concentration of Mg 2ϩ is 1-3 mM (23), the lower, 5 mM concentration more closely mimics physiological conditions. For decades, it has been known that magnesium ions bind ribosomal subunits (24 -29) through interactions with the negatively charged rRNA backbone and, in this manner, facilitate structural alterations in the ribosome (24). Thus, it is easy to envision that high Mg 2ϩ concentrations in cell lysis/gradient buffers might alter the pre-60 S subunit in such a way that the Arx1-pre-60 S subunit interaction is destabilized.…”

Section: Discussionmentioning

confidence: 99%

The Cytosolic J-protein, Jjj1, and Rei1 Function in the Removal of the Pre-60 S Subunit Factor Arx1

Meyer¹,

Hoover

Craig

2010

Journal of Biological Chemistry

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Although the biogenesis of ribosomal subunits occurs predominantly in the nucleus, final remodeling steps take place in the cytosol. One cytosolic step has two components: 1) the removal of the maturation factor Arx1, which transits from the nucleus to the cytosol with the pre-60 S subunit, and 2) its subsequent transport back into the nucleus. Two cytosolic proteins, Rei1 and Jjj1, are required, but their individual contributions to this step are not understood. Here we report that Rei1 and Jjj1 directly interact. This interaction is mediated by a C-terminal segment of Jjj1 encompassing a region rich in charged residues, flanked by C 2 H 2 -type zinc fingers. Deletion of the charged region results in defects in 60 S subunit biogenesis in vivo. In addition, we report resolution of an apparent contradiction in the literature regarding the association of Arx1 with the pre-60 S subunit in the absence of Rei1. The association of Arx1 with ribosomes is sensitive to the concentration of magnesium ions when Rei1 is absent. At near physiological concentrations, Arx1 remains associated with the pre-60 S particle, as it does in the absence of Jjj1; at higher concentrations, Arx1 dissociates in the absence of Rei1 but not in the absence of Jjj1. As both Rei1 and Jjj1 are required for dissociation of Arx1 from the pre-60 S subunit, and the region of Jjj1 that mediates interaction with Rei1 is required in vivo for 60 S subunit biogenesis, our data support the idea that the primary role of both Rei1 and Jjj1 is the first step of the Arx1 removal/recycling process.

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“…Still, these results must be further clarified by cryo-EM and smFRET analyses, as the FRET state changes could reflect motions of IRES RNA, the ribosome and/or the SNAP-tag label on eS25. Curiously, however, both the addition of translation extract and reduced Mg 2þ concentration (2.5 versus 5 mM) funneled the 40S : IRES complexes into a single conformation, suggesting that the 40S : IRES conformational dynamics are sensitive to their chemical environment [135].…”

Section: (G) Dynamic Rearrangements Within the 80s : Ires Complexmentioning

confidence: 99%

Dynamics of IRES-mediated translation

Johnson

Grosely

Petrov

et al. 2017

Phil. Trans. R. Soc. B

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One contribution of 12 to a theme issue 'Perspectives on the ribosome'. Viral internal ribosome entry sites (IRESs) are unique RNA elements, which use stable and dynamic RNA structures to recruit ribosomes and drive protein synthesis. IRESs overcome the high complexity of the canonical eukaryotic translation initiation pathway, often functioning with a limited set of eukaryotic initiation factors. The simplest types of IRESs are typified by the cricket paralysis virus intergenic region (CrPV IGR) and hepatitis C virus (HCV) IRESs, both of which independently form high-affinity complexes with the small (40S) ribosomal subunit and bypass the molecular processes of cap-binding and scanning. Owing to their simplicity and ribosomal affinity, the CrPV and HCV IRES have been important models for structural and functional studies of the eukaryotic ribosome during initiation, serving as excellent targets for recent technological breakthroughs in cryogenic electron microscopy (cryo-EM) and single-molecule analysis. High-resolution structural models of ribosome : IRES complexes, coupled with dynamics studies, have clarified decades of biochemical research and provided an outline of the conformational and compositional trajectory of the ribosome during initiation. Here we review recent progress in the study of HCV-and CrPV-type IRESs, highlighting important structural and dynamics insights and the synergy between cryo-EM and single-molecule studies.This article is part of the themed issue 'Perspectives on the ribosome'.

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Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

Cited by 50 publications

References 67 publications

Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site

Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site

The Cytosolic J-protein, Jjj1, and Rei1 Function in the Removal of the Pre-60 S Subunit Factor Arx1

Dynamics of IRES-mediated translation

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