Abstract

ABSTRACT In bacteria, many genes involved in the biosynthesis of cofactors such as TPP are regulated by riboswitches, a part of an mRNA molecule that can directly bind a small target molecule. Upon binding, these RNA elements undergo conformational changes which then affect the gene’s activity. In the green alga Chlamydomonas reinhardtii, we have found that TPP riboswitches are found in THI4 and THIC , the genes encoding the first enzymes of the thiazole and pyrimidine branches respectively of the thiamine biosynthesis pathway. The transcripts of these genes are found to be alternatively spliced when thiamine is added to the cultures and it is suggested that the presence of the riboswitches may play a role in this (Croft et al., 2007). The focus of my research has been to study how thiamine biosynthesis is regulated in C. reinhardtii mutants, especially the pyr1 mutant. The pyr1 mutant of C. reinhardtii (Smyth et al., 1975), which is resistant to the thiamine analogue pyrithiamine, has a mutation in the THI4 riboswitch that prevents the THI4 gene from being repressed by TPP . I have analysed the growth and expression of thiamine biosynthesis genes in wild type and C. reinhardtii pyr1 mutants in the presence of thiamine and pyrithiamine. Using a construct in which the THI4 riboswitch is cloned upstream of the luciferase gene, I have carried out in vivo expression analysis of the effect of mutations in conserved bases located in P2 and P4 stems of the C. reinhardtii THI4 riboswitch. I observed high expression of luciferase activity without the presence of thiamine, and low expression with the presence of thiamine in the non-mutated riboswitch. When a single base located in the P2 stem was changed to mimic the pyr1 mutant, the luciferase activity was maintained even in the presence of thiamine. A single base change of one the conserved residues in the P4 stem also resulted in a stable luciferase activity in the presence of thiamine. The results confirm the participation of bases located in P2 and P4 stems in the recognition of TPP as well as the consequence of binding of TPP towards the expression of THI4 gene in C. reinhardtii.