Mentor: Dr. Susan White
RNA, a medium for the communication of genetic information in a cell, plays various roles which may be better understood by the elucidation of its 3-D secondary structure, i.e. how its individual nucleotides are arranged. RNA secondary structure is complex, similar to the 3-D folding of a protein, consisting of motifs such as hairpins, pseudoknots, etc. This summer’s project involves a study of the thermodynamics of the secondary structure of L30 RNA. The L30 ribosomal protein in Saccharomyces cerevisiae suppresses its own splicing and translation by binding to its messenger RNA. The kink-turn (see figure 1) is a structural motif that causes a sharp bend in a RNA double-helix and is present both in L30 messenger and ribosomal RNA.
This is thought to play an important role in the binding of L30 to its RNA.
In vitro synthesis of L30 DNA (wild type and mutant) will be carried out with subsequent transcription to make RNA. The different RNA transcripts will then be subjected to Tm analysis, i.e. thermal denaturation profiles will be constructed to obtain melting temperatures, i.e. the temperature at which the RNA unfolds, with the purpose of determining the effect of changing certain base pairs on the Tm (melting temperature) and the ?G and ?H values. It is expected that changing certain bases will lower the melting temperature. Thus, the role played by various bases in the formation of the kink-turn will be elucidated. Hence, it is hoped to increase our understanding of the process of manufacture of the ribosomal protein L30 and, the formation of ribosomes.
Figure1. Kink turn in L30 RNA. The boxes indicate nucleotides A, C, G and U. The nucleotides in green are the stems, the ones in royal blue are stacked on the stems, and the orange ones form the kink turn.