The effect of a single base change in THI4 riboswitch on TPP binding in C. reinhardtii
Zetty Norhana Balia Yusof, Michael Moulin and Alison G. Smith
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
In bacteria, many genes involved in the biosynthesis of cofactors such as TPP are regulated by riboswitches. Riboswitches are genetic control elements located mainly within the 5' untranslated regions of messenger RNAs. Upon binding of regulatory signals, which include vitamins, amino acids, nucleobases and uncharged tRNA, these RNA elements undergo conformational changes that modulate gene expression. The thiamin pyrophosphate (TPP)-binding riboswitch is found in all three kingdoms of life and can regulate gene expression via premature termination of transcription, initiation of translation and mRNA splicing. 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. In this study we report an in vivo expression analysis of the effect of mutations in conserved bases located in P2 and P4 stems of the C. reinhardtii THI4 riboswitch. We performed an analysis of mutants with single base substitutions in the riboswitch linked with a luciferase gene and observed the expression with the absence or presence of thiamin. We observed high expression of luciferase activity without the presence of thiamin and low expression with the presence of thiamin in the non-mutated riboswitch. When a single base located in the P2 stem was changed to mimic the pyr1 mutant (pyrithiamine resistant 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.
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