A IRES (internal ribosome entry site) is a nucleotide sequence that allows for the translation initiation in the middle of a message RNA sequence as part of the greater process of protein synthesis. Typically an IRES is located in the 5’ UTR of RNA viruses and enables translation of the RNAs in a cap-independent manner. (1,2)
Evaluating a particular RNA sequence for IRES activity relies on designing a bicistronic report construct. When an IRES segment is located between two reporter gene (e.g., Chloramphenicol acetyltransferase [CAT] and Luciferase) open reading frames, it can drive translation of the downstream protein coding region independently from the 5´ cap structure bound to the 5´ end of the mRNA (3,4,5).
By utilizing this experimental design specific IRES domains can be mapped either by creating a series of deletion or mutation constructs. Small molecules that specifically inhibition virus directed translation can also be screened for using this technology. Both cell based and cell free expression systems can be used to conduct these experiments.
When using a CAT/Luciferase constructs in a cell based expression system, cell extracts are prepared and results are based on detection by Western blotting using antibodies against each reporter. When using cell free based expression systems incorporation of 35Met followed by gel analysis is the standard method for detection..
By using a Renilla Luciferase/FireFly Luciferase construct (6), time consuming immunoblotting and radioactivity can be avoided. Detection of both reporters can accomplish by using a single assay system (e.g., Dual-Glo™ Luciferase Assay System).
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