Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3 ^′ UTR of turnip crinkle virus

Abstract

The 3 0 untranslated region (3 0 UTR) of turnip crinkle virus (TCV) genomic RNA contains a cap-independent translation element (CITE), which includes a ribosome-binding structural element (RBSE) that participates in recruitment of the large ribosomal subunit. In addition, a large symmetric loop in the RBSE plays a key role in coordinating the incompatible processes of viral translation and replication, which require enzyme progression in opposite directions on the viral template. To understand the structural basis for the large ribosomal subunit recruitment and the intricate interplay among different parts of the molecule, we determined the global structure of the 102-nt RBSE RNA using solution NMR and small-angle x-ray scattering. This RNA has many structural features that resemble those of a tRNA in solution. The hairpins H1 and H2, linked by a 7-nucleotide linker, form the upper part of RBSE and hairpin H3 is relatively independent from the rest of the structure and is accessible to interactions. This global structure provides insights into the three-dimensional layout for ribosome binding, which may serve as a structural basis for its involvement in recruitment of the large ribosomal subunit and the switch between viral translation and replication. The experimentally determined threedimensional structure of a functional element in the 3 0 UTR of an RNA from any organism has not been previously reported. The RBSE structure represents a prototype structure of a new class of RNA structural elements involved in viral translation/replication processes.new method | NMR | SAXS | 3' UTR RNA | RNA structure S tructural elements in mRNAs such as the 5 0 cap, internal ribosome entry site in the 5 0 UTR, and the 3 0 poly(A) tail in the 3 0 UTR are important determinants for efficient translation initiation (1). These structural elements can function synergistically to attract ribosomes and translation factors to enhance translation initiation (2, 3). In both cap and poly(A) tail-dependent translation in eukaryotes this enhancement is realized by the binding factor elF4G associating with both the polyA binding protein (Pab1p) and elF4E, resulting in a circularized mRNA template (4), which has been visualized under atomic force microscopy (5). In contrast, high level initiation of capindependent translation in many plant viruses involves 3 0 UTR RNA elements known as CITE (6). TCV lacks a 5 0 cap and poly(A) tail. Instead the virus uses a structural element in its 3 0 UTR that synergistically enhances translation when associated with its 5 0 UTR (7) (Fig. 1A). A model for 3 0 UTR involvement in the recruitment of the large ribosomal subunit has been proposed to account for the enhancement (7) but the experimental structural basis for such an involvement has not been demonstrated. Moreover, mechanisms are required to temporally coordinate viral replication and translation because these processes are mutually exclusive due to the opposing directions of protein and RNA synthesis. The infecting genomic RNA must first be trans...

Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase

“…5B), EcRapA-TtCore, and EcRapA⌬N-TtCore (Fig. 6B) are also in good agreement with those calculated from experimental data as judged on the basis of previous studies (36,37). Furthermore, we show here that SAXS can be used to complete large, multisubunit structures with missing components.…”

Translational Regulation of Cytoplasmic mRNAs

“…Many utilize cap-independent translational enhancer elements in the 3' UTR or in the 5' UTR. While cellular mRNAs are thought to form a closed loop via the PABP-eIF4G bridge, turnip crinkle virus attracts the 60S ribosomal subunit via a tRNA like structure in the 3' UTR, which then basepairs with a translational enhancer in the 5' UTR to stimulate translation (Stupina et al, 2008;Zuo et al, 2010;Stupina et al, 2011). It will be interesting to discover whether similar mechanisms are utilized by plant encoded mRNAs.…”

Structure of the yeast U2/U6 snRNA complex

“…The resulting perturbed models were sorted again as described above. The selected models were then refined against SAXS data, RDC measurements, and base-pairing restraints in XPLOR-NIH as previously described (Zuo et al 2010). Distance restraints based on the previously determined U6 ISL structure (Venditti et al 2009) were incorporated on the basis of chemical shift similarity between the U6 ISL construct and the 111-nt U2/U6 construct.…”

“…Comparison of the predicted scattering curves of the states obtained from NMA with experimental SAXS data resulted in reselection of the original states, leading to the conclusion that the originally selected models reflect the true ground-state conformation of U2/U6 (data not shown). Finally, the 10 structural models were refined simultaneously against SAXS and RDC measurements using restrained molecular dynamics and energy minimization in XPLOR-NIH as previously described (Zuo et al 2010).…”