Tiffany Sheng

 

Comparison of the L30e Protein in Yeast to the Analogous Protein in Humans Using Site-directed Mutagenesis

 

Mentor: Dr. Susan A. White

 

 

ABSTRACT

 

Yeast ribosomal protein L30e has been shown to bind to its own transcript to regulate the splicing and translation of its own gene. The protein binds to the region in RNA known as the kink-turn motif. In recent experiments, the human L30e protein has been crystallized and its atomic level structure has been solved by X-ray diffraction.

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Figure 1. Cartoon representation of the human L30e protein. The sequence identity between human L30e and yeast L30e was shown to be 53.9%. Intramolecular interactions, which are shown in stick representation and dashed hydrogen bonds, are not present in yeast L30e. (Kawaguchi et al., 2011). PDB Reference: ribosomal protein L30e, 3vi6.

 

When comparing the structure of the L30e protein in yeast and humans, it was observed that the yeast protein lacked cysteine, an amino acid that is present in the human counterpart. Using site-directed mutagenesis, our goal is to perform analogous substitution in regions of the yeast DNA relevant to protein binding. In this region, specific bases in the DNA sequence will be altered to correspond to the codon for cysteine. The purpose of this experiment is to determine whether or not the L30e protein still binds to the mutated transcript. In addition, because cysteine is one of the few amino acids containing sulfur, this can also allow for the attachment of other fluorescent probes.

 

References

Kawaguchi, A., Ose, T., Yao, M., and Tanaka, I. (2011). Acta Crystallographica Section F Structural Biology and Crystallization Communications. F67, 1516-1518.