Internal ribosomal entry sites (IRES) are an important but poorly understood part of the eukaryotic translational machinery, allowing cap-independent translation initiation. Normally, translation initiation requires the binding of eukaryotic initiation factor 45 (eIF45) to a 7-methyl guanosine cap at the 5’ end of the RNA. This initiation factor recruits the ribosome, which then scans for the first AUG codon and begins translation of the open reading frame. However, many cellular proteins are either positioned as the second open reading frame in a bicistronic strand of mRNA, or contain highly structured 5’ untranslated regions, preventing efficient scanning from the 5’ end. This presents an interesting challenge for synthetic biologists, as it prevents the engineering of multicistronic operons that contain two or more genes under the control of the same promoter. Luckily, nature has come up with its own remedy for this problem, known as internal ribosomal entry site (IRES) mediated translation initiation, which allows the ribosome to bind in the middle of a strand of mRNA and initiate translation. In essence, an IRES is a highly structured region of untranslated mRNA that recruits eukaryotic initiation factors to begin translation initiation.
Unfortunately, the Registry of Standard Biological Parts contains few IRESs and even those are poorly annotated. The UTK-Knoxville iGEM team hopes to begin to remedy this problem by characterizing a group of IRESs and submitting them to the registry. In addition, as a part of our characterization efforts, we plan to develop a methodology of determining relative IRES strength, as modeled by the methodology put forth by Kelly et al in the Journal of Biological Engineering (Kelly et al., 2001). Using flow cytometry, we will determine the relative levels of fluorescent protein under the control of each IRES, as expressed in S. cerevisiae. Finally, we will show a practical example of how IRESs can be used in chemical engineering and synthetic biology. This work will not only be useful for other projects within our sponsor lab, but will also serve the greater synthetic biology community by initiating the growth of a library of IRESs and a protocol to allow contribution by all members of the community.
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