Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stemloop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.optical tweezers ͉ RNA triplexes ͉ single-molecule ͉ RNA folding M essenger RNAs (mRNAs) designate proteins by sequences of codons consisting of 3 nucleotides each. The reading frame is defined by a start codon (AUG) and is usually maintained by ribosomes (with an error rate less than 3 ϫ 10 Ϫ5 ) (1). Programmed -1 ribosomal frameshifting (FS) has been found to express defined ratios of structural and enzymatic proteins in many viruses including HIV (2-6). Programmed FS has also been found during expression of cellular genes (6-8). Highly efficient FS at an mRNA slippery sequence from X XXY YYZ (0 frame) to XXX YYY Z (-1 frame) is often stimulated by a downstream pseudoknot structure (Fig. 1A). X can be any 3 identical nucleotides, Y can be either AAA or UUU, and Z is usually not G (6, 9-11). The slippery sequence and pseudoknot structure are typically separated by a single-stranded linker of 5-10 nucleotides. The natural high-efficiency FS stimulatory structure is often a hairpin (H)-type pseudoknot, which involves base pairing between nucleotides in a hairpin loop and nucleotides outside of the hairpin loop. It has been shown that tertiary minor-groove interactions between stem 1 and loop 2 (base triples) of pseudoknots are critical for programmed FS in certain viruses (12-16).All of the secondary and tertiary structures in coding regions of mRNA have to be unfolded for translation. With the mRNA slippery sequence at the aminoacyl (A) and peptidyl (P) sites of the ribosome, the downstream pseudoknot structure is believed to be in contact with the helicase domain of the ribosome and provides mechanical resistance to ribosomal translocation (17-23). The detailed molecu...

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The double‐stranded RNA‐binding motif (dsRBM) is an αβββα fold with a well‐characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM‐containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A‐form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non‐RNA targets (proteins and DNA), and act in combination with other dsRBMs and non‐dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM–protein recognition.

Base-pair formation between two hairpin loops-a "kissing" complex-is an RNA-folding motif that links two elements of RNA secondary strcture. It is also a unique protein recognition site involved in regulation of CoIEl plasmid DNA replication. The trans-activation response element (TAR), a hairpin and bulge at the 5' end of the untrans-

RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3′ direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) −1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate−1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for−1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing−1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study’s findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.