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The intriguing feature of picornavirus IRES elements of type I is the presence of conserved upstream AUG codon. In case of poliovirus IRES, this putative initiation codon is located 157 nt upstream of the main AUG and is out of frame to the poliovirus genome reading frame. However, it is known that mutation of this AUG result in inhibition, rather than stimulation of translation of the main ORF. Here we investigated the effect of Glycyl tRNA synthetase (GARS), the recently identified crucial ITAF required for picornavirus type I translation, on translation initiation site selection. Surprisingly, GARS stimulates translation more efficiently when reporter was fused directly with uAUG than for wt PV IRES. When uAUG was mutated the effect of GARS becomes less prominent. Thus, GARS mediates loading of ribosomes to uAUG which in turn results in increase of initiation on downstream main AUG. In order to understand how GARS activates translation of PV IRES, cell free translation system was supplemented with various translation initiation factors with or without GARS. eIF4G fragment p100 was able to compensate the absence of GARS added. The shorter fragment of eIF4G (eIF4G MD) was also able to stimulate translation from main AUG, but unexpectedly was strongly inhibitory to translation driven from uAUG. We propose that GARS increases binding of eIF4G to PV IRES and regulates eIF4G activity in order to allow the initiation on uAUG followed by transfer of ribosome to the main AUG codon.