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Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G+3 in the mRNA. In such pairs the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.
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Structural characterization of a new subclass of panicum mosaic virus-like 3′ cap-independent translation enhancer
Abstract Canonical eukaryotic mRNA translation requires 5′cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5′cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3′UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson–Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson–Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.
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- Award ID(s):
- 1818229
- PAR ID:
- 10406997
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 3
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 1601 to 1619
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3′ structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3′ cap-independent translation enhancers (3′CITEs), as umbraviruses can have different 3′CITEs in the central region of their lengthy 3′UTRs, and most also have a particular 3′CITE (the T-shaped structure or 3′TSS) near their 3′ ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3′CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3′CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3′CITEs that differentially affect the structure and translation of different umbraviruses.more » « less
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It has been shown previously in protonated, deprotonated and ionized guanine–cytosine base pairs that intra-base pair proton transfer from the N1–H at the Watson–Crick edge of guanine to the complementary nucleobase prompts non-statistical dissociation of the base-pair system, and the dissociation of a proton-transferred base-pair structure is kinetically more favored than that of the starting, conventional base-pair structure. However, the fundamental chemistry underlying this anomalous and intriguing kinetics has not been completely revealed, which warrants the examination of more base-pair systems in different structural contexts in order to derive a generalized base-pair structure–kinetics correlation. The purpose of the present work is to expand the investigation to the non-canonical homodimeric and heterodimeric radical cations of 9-methylguanine (9MG) and 9-methyl-8-oxoguanine (9MOG), i.e. , [9MG·9MG]˙ + , [9MOG·9MG]˙ + and [9MOG·9MOG]˙ + . Experimentally, collision-induced dissociation tandem mass spectrometry coupled with an electrospray ionization (ESI) source was used for the formation of base-pair radical cations, followed by detection of dissociation product ions and cross sections in the collisions with Xe gas under single ion–molecule collision conditions and as a function of the center-of-mass collision energy. Computationally, density functional theory and coupled cluster theory were used to calculate and identify probable base-pair structures and intra-base pair proton transfer and hydrogen transfer reactions, followed by kinetics modeling to explore the properties of dissociation transition states and kinetic factors. The significance of this work is twofold: it provides insight into base-pair opening kinetics in three biologically-important, non-canonical systems upon oxidative and ionization damage; and it links non-statistical dissociation to intra-base pair proton-transfer originating from the N1–H at the Watson–Crick edge of 8-oxoguanine, enhancing understanding towards the base-pair fragmentation assisted by proton transfer.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1093/nar/gkac007
