Artificial OFF-Riboswitches That Downregulate Internal Ribosome Entry without Hybridization Switches in a Eukaryotic Cell-Free Translation System

The RNA genome of Plautia stali intestine virus (PSIV; Cripavirus, Dicistroviridae) contains two open reading frames, the first of which is preceded by a 570 nt untranslated region (5′ UTR). The 5′ UTR was confirmed to be an internal ribosome entry site (IRES) using an insect cell lysate translation system: translation of a second cistron increased 14-fold in the presence of the 5′ UTR and a cap analogue did not inhibit translation of the second cistron. Deletion analysis showed that 349 bases corresponding to nt 225–573 in the PSIV genome were necessary for internal initiation. The PSIV 5′ IRES did not function in rabbit reticulocyte lysate or wheatgerm translation systems; however, the intergenic IRES for capsid translation of PSIV was functional in both systems, indicating that the 5′ IRES and the intergenic IRES have distinct requirements for their activities. Chemical and enzymic analyses of the 5′ IRES of PSIV indicate that its structure is distinct from that of Rhopalosiphum padi virus. Because 5′ IRES elements in some dicistroviruses have been reported to be active in plant and mammalian cell-free translation systems, there appears to be variation among dicistroviruses in the mechanism of translation initiation mediated by 5′ IRES elements.

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Protein secondary structure determines the temporal relationship between folding and disulfide formation

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011983 ◽

2020 ◽

Vol 295 (8) ◽

pp. 2438-2448 ◽

Cited By ~ 5

Author(s):

Philip J. Robinson ◽

Shingo Kanemura ◽

Xiaofei Cao ◽

Neil J. Bulleid

Keyword(s):

Secondary Structure ◽

Cell Biology ◽

Disulfide Bonds ◽

Protein Secondary Structure ◽

Eukaryotic Cell ◽

Translation System ◽

Disulfide Formation ◽

Free Translation ◽

Cellular Context ◽

Disintegrin Domain

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

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