stem-loop. These modifications affect other aspects of translation such as the ability of the ribosome to maintain the three-nucleotide codon of the mRNA as it moves through the ribosome. The absolute requirement for precise correlation between the mRNA frame and the correct protein sequence to be expressed underlies an fundamental question in molecular biology: what regulates the mRNA reading frame? To address this question, we study defined biological systems that subvert the three-nucleotide mRNA reading frame resulting in high levels of frameshifting. Our biochemical and structural results reveal that tRNA distortion and conformational changes of the small ribosomal subunit are induced by frameshift-prone tRNAs. This dysregulation causes the ribosome to lose its grip on the mRNA, allowing the tRNA to shift into a new reading frame. Together these studies reveal how the ribosome undergoes recoding into alternative mRNA frames and suggests how dysregulation of the mRNA frame disrupts key interactions between tRNAs, mRNA, and translation factors with the ribosome.
A precolumn derivatization liquid chromatography (LC) method was developed for the analysis of various dietary supplement formulations and raw materials for glucosamine. A 1 mL sample or standard water solution (containing about 0.05 mg glucosamine) was mixed with 1 mL pH 8.3 buffer, 1 mL 5% phenylisothiocyanate methanolic solution, and derivatized at 80°C in a water bath for 30 min. After derivatization, the solution was cooled in a cold water bath and centrifuged at 3000–5000 rpm. The clear upper layer was ready for injection. The LC system was equipped with a C18 reversed-phase column and an ultraviolet detector set at 240 nm. The column was developed with a linear gradient composed of 0.1% phosphoric acid in deionized water and 0.1% phosphoric acid in methanol. The method was subjected to Single Laboratory Validation. The method precision was 0.50% relative standard deviation, accuracy was less than ±1.5%, method linearity in the range 0–2 mg glucosamine/mL was 1.00, the detection limit was 0.0705 μg/mL, and the quantitation limit was 0.235 μg/mL. Chondroitin sulfate, amino acids, and excipients did not interfere with glucosamine testing. After derivatization, both standard and sample preparations were stable for at least 48 h. Due to its high sensitivity, this method can be used to assay glucosamine in functional foods and pet foods. The validation data will be published separately.
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