PHYSICAL STUDIES OF RNA STRUCTURAL MOTIFS

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RNA molecules are active participants in many cellular processes, yet little is known about fundamental aspects of their structure, dynamics, or folding mechanisms; only transfer RNAs are understood in detail. Four different fragments of larger RNAs have been identified as containing interesting structure relevant to their biological roles and potentially illustrative of strategies used by larger RNAs to achieve specific functions. They are: i. A complex mRNA pseudoknot serves as a regulatory site for a translational repressor; it has substantial tertiary structure in addition to the known secondary structure, must unfold and refold during translation, and probably adopts alternative conformations. ii. A conserved rRNA hairpin with bulge loop shows competition between a pseudoknot structure formed by the bulge and hairpin loops, and a structure within the hairpin loop. iii. An mRNA pseudoknot from a retrovirus causes readthrough of stop codons; sequences 5' to the structure and within one loop may form additional structure. iv. A junction of three helices from the large subunit ribosomal RNA contains substantial tertiary structure that is stabilized by specific binding of single Mg2+ and NH4+ ions. Several experimental approaches will be used to elucidate the thermodynamics of folding tertiary structure in each of these, and to further explore the kinetics of folding and the roles of specific and non- specific cations. Particular attention will be paid to "conformational switches", which may be a common feature of RNA function. In a set of collaborative experiments, transient electric birefringence will be used to determine the persistence length and rinse per base pair of random sequence duplex RNA in solution, and investigate the effects of bulge loops on RNA bending and flexibility.