Michael P. Weir, Joe Coolon, Amy MacQueen
Translation is the fundamental process through which proteins are produced in cells and organisms. The process occurs with three main components: mRNA, tRNAs, and the ribosome and while translation mechanisms are generally well understood, our knowledge far from complete. Previous work from the Weir laboratory has analyzed a 3-nucleotide periodicity in mRNA ORFs downstream of the start codon that is characterized by “ GCN” with significant G-depression at position 2 of codons. This periodicity is enhanced for proteins with high expression levels. A highly conserved 530 loop 16S/18S rRNA sequence, located in the mRNA entry tunnel of the ribosomal small subunit, was found previously to have complementarity to the mRNA periodicity. Structural analyses of high-resolution ribosome structures have informed our hypothesis that potential transient hydrogen bonding interactions between the 530 loop and mRNA nucleotides may contribute to modulating protein expression. Our analysis presents two models of interaction between the mRNA and the 530 loop during translocation steps. The first is the 2-mer model which proposes interaction between bases G530 and G529 with the mRNA. The second model is the 4-mer model which hypothesizes interaction between bases G530, G529, C528, and G527 with the mRNA. In this work, we test our hypothesis through mutational analysis of two candidate genes. We created mutants downstream of their translation start sites using CRISPRCas9 gene editing and performed protein expression analysis using western blot analysis. Expression studies of two of our mutant genes has suggested that disrupting normal phasing of the aligned mRNA and 530 loop sequence suppresses translations and that increased in-phase base pairing increases fidelity of start site choice but dampens protein expression. These observations are encouraging and with further testing of additional mutants, this research may help to increase our knowledge of translation mechanisms.
Sheth, Ruchi B., "A Mutational Analysis of Open Reading Frame Periodicity" (2017). Masters Theses. 159.
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