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Tinsley, Melissa Marie

Microbiology and Molecular Genetics

2007

MS

The importance of RNA continues to grow as research reveals its diverse roles within the cell. Like DNA, RNA possesses the genetic code. Yet RNA's responsibilities far exceed that of DNA. RNA serves as an intermediary relating messages between DNA and proteins, but has also been shown to function on multiple levels of gene regulation and has also been shown to possess enzymatic activity. Therefore it is not hard to imagine a primitive environment where RNA was responsible for carrying out the functions of life. To carry out such tasks as gene regulation and enzymatic activity, without the help of proteins, RNA must utilize ions and metabolites within its environment. Riboswitches are RNA elements that regulate gene expression through structural rearrangements usually upon ligand binding. These conformational changes can cause attenuation of transcription by formation of hairpin structures, or can cause inhibition of translation by sequestering the ribosome-binding site. One riboswitch, known as the glmS RNA element, possess enzymatic activity and as such acts primarily as a ribozyme. Unlike other riboswitches it relies on cations rather than ligand binding to adopt its active fold. This is reminiscent of the other ribozymes, which rely heavily on cations for tertiary structure and in some cases catalytic activity. RNA must undergo complex folds, similar to that of proteins when serving as a ribozyme. In some cases, such as group I introns and the hairpin ribozyme, non-catalytically essential accessory elements allow the ribozyme to form a compact structure capable of catalysis under physiological concentrations of ions, such as magnesium. This thesis will focus on deciphering the role of highly conserved, non-essential elements 3' to the catalytic core of the glmS ribozyme-riboswitch. Based on kinetic analysis and hydroxyl radical footprinting experiments, it is believed that these accessory domains help the ribozyme to fold and carry out catalysis at cellular levels of magnesium.