Difference between revisions of "Part:BBa K1993016"

Latest revision as of 19:13, 17 October 2016

IRES

IInternal ribosome entry site (IRES) is a mRNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex. The location for IRES elements is often in the 5'UTR, but can also occur elsewhere in mRNAs.

IRES sequences were first discovered in 1988 and encephalomyocarditis virus (EMCV) RNA genomes [1]. It is used by viruses as a means to ensure that viral translation is active when host translation is inhibited. But to date, when an IRES segment is located between two reporter open reading frames in a eukaryotic mRNA molecule (a bicistronic mRNA), it can drive translation of the downstream protein coding region independently of the 5'-cap structure bound to the 5' end of the mRNA molecule. In such a setup both proteins are produced in the cell. The first reporter protein located in the first cistron is synthesized by the cap-dependent initiation, while translation initiation of the second protein is directed by the IRES element located in the intercistronic spacer between the two reporter protein coding regions.

IRES has been widely used due to the following advantages: (1) ensured coexpression of genes before and after the IRES; (2) feasibility of adding subcellular localization sequences to the gene after IRES; and (3) availability of commercial expression plasmids harboring IRES. [2]

In our project, firstly we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Then we acquired it from a plasmid we purchased before.(Figure 1). Taking advantage of function of IRES, we constructed a plasmid that IRES linked between two protein coding genes. For example, we constructed BBa_K1993005 (Luciferase-IRES-eGFP) to ensure eGFP could be expressed.

Figure 1 Purification of IRES.

References

[1] Jang SK, Kräusslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E (August 1988). "A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation". J. Virol. 62 (8): 2636–43

[2] Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. [J]. Plos One, 2011, 6(4): e18556.

Sequence and Features