Difference between revisions of "Part:BBa K1442023"

Revision as of 14:05, 7 October 2014

EMCV IRES

This is an Internal Ribosome Entry Site (IRES) acting in mammalian cells. It allows translation initiation in the middle of an RNA strand which is required for the fuctioning of our replicon as there are multiple protein coding regions separated by structural nucleotide sequences interspersed throuhgout the RNA strand. We chose to investigate two IRES sequences to compare the relative efficiency and determine which was better for incorporation into our system. These were the Encephalomyocarditis virus (EMCV) and the IRES derived from the NF-kappaB repressor factor (NKRF) untranslated region.

IRES' were first discovered in EMCV and polioviruses and the EMCV has been the favoured IRES for use in replicon systems such as Lohmann (1999) and Lee (2005).

IRESes are commonly used by viruses to ensure translation is active even during times when host translation is inhibited. This is ideal for our system as in order to maintain the RNA in the cell for the maximum length of time the translation of the RdRp should be constant to balance the instability and subsequent degradation of the RNA strands.

The EMCV IRES functions via its secondary structure, a complex series of hairpin loops and double stranded sections including a bifarcation loop, which attracts ribosomes to the vicinity of the start codon of the protein coding region of the RNA.

The secondary structure of the EMCV IRES predicted using Vienna RNAfold calculating minimum free energy of nucleotide pairing

This is detailed in the Bochkov and Palmenberg (September 2006) paper on translational efficiency of EMCV IRES and details the minimum and preferred regions included for functional translational action.

We used the extended preferred region referenced in this image, in order to optimize the efficacy of this subunit of the replicon.

Usage

This IRES was intended for used before the neomycin selectivity marker and the NS5B RdRp derived from the HCV strain 1b genome, as both of these proteins are integrated into the RNA strand with structural nucleotide sequences before and after.

This is a schematic of the entire replicon and indicating the positioning of the IRESes

Diagram generated by Genious.

part BBa_K1442109

This testing module was used to determine the efficacy of the IRESes by measuring the fluorescence in human HeLa and Huh7.5 cells using a tecan plate reader. The IRES was cloned between the AscI and BsrGI site following the T7 promoter and before the GFP. This was sequenced to ensure no mutations during the PCR amplification of the parts synthesised by IDT.