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...

Background:Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species – P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116–125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12–73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

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.

An efficient −1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote −1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several −1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of −1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the −1 PRF efficiency of this stimulator can be modulated by sequential removal of base–triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem–loop interactions and their relative positions to the helical junction play crucial roles for the −1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate −1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate −1 PRF. Furthermore, it suggests that −1 PRF, induced by an in-trans RNA via specific base–triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.

Specific recognition of metabolites by functional RNA motifs within mRNAs has emerged as a crucial regulatory strategy for feedback control of biochemical reactions. Such riboswitches have been demonstrated to regulate different gene expression processes, including transcriptional termination and translational initiation in prokaryotic cells, as well as splicing in eukaryotic cells. The regulatory process is usually mediated by modulating the accessibility of specific sequence information of the expression platforms via metabolite-induced RNA conformational rearrangement. In eukaryotic systems, viral and the more limited number of cellular decoding À1 programmed ribosomal frameshifting (PRF) are commonly promoted by a 39 mRNA pseudoknot. In addition, such À1 PRF is generally constitutive rather than being regulatory, and usually results in a fixed ratio of products. We report here an RNA pseudoknot capable of stimulating À1 PRF whose efficiency can be tuned in response to the concentration of S-adenosylhomocysteine (SAH), and the improvement of its frameshifting efficiency by RNA engineering. In addition to providing an alternative approach for small-molecule regulation of gene expression in eukaryotic cells, such a metabolite-responsive pseudoknot suggests a plausible mechanism for metabolite-driven translational regulation of gene expression in eukaryotic systems.