| Popis: |
Eukaryotic Initiation Factor (eIF) 4A is the most abundant initiation factor and the prototypical member of the DEAD-box family of helicases. Once recruited to the cap-binding complex, eIF4F, eIF4A unwinds inhibitory RNA secondary structure in the 5’ untranslated region (UTR) of mRNAs, promoting efficient ribosomal scanning to the start codon. The requirement for eIF4A in translation initiation correlates with increasing 5’ UTR length, suggesting that regulating the activity of eIF4A may affect the translation of particular mRNAs. It is well established that the transcripts of genes involved in cell cycle control and proliferation have long 5’ UTRs; therefore altering the activity of eIF4A may affect these genes specifically. A cross-discipline approach was used to investigate eIF4A helicase activity to obtain information regarding both the mechanics of helicase activity and the biological impacts of its inhibition. Recombinant eIF4A helicase activity, the stimulatory effect of eIF4B and the effect of known eIF4A inhibitors was first analysed using an ensemble helicase assay. Due to the limitations of this method a single molecule technique utilising optical tweezers was developed to investigate helicase activity at a higher force resolution. Optical tweezers were used to ‘trap’ and manipulate a dual-labeled RNA:DNA construct containing a central stem-loop hairpin known to be inhibitory to ribosomal scanning attached to functionalised microspheres. Although instrumental failure prevented the completion of these experiments, initial force extension curves using this molecule were obtained. Once established, this single molecule system may be used to observe eIF4A activity with its accessory protein eIF4B and known eIF4A inhibitors. 15-deoxy-delta(12, 14)-prostaglandin J2 (15d-PGJ2) is a newly identified natural inhibitor of eIF4A activity which induces apoptosis and is implicated in the resolution of inflammation. The translation of specific mRNAs affected by 15d-PGJ2 treatment of HeLa cells was analysed by translational profiling coupled to microarray analysis. No correlation, however, was seen between those transcripts that were translationally repressed and their 5’ UTR length or composition. |
