The role of eukaryotic initiation factor 4F in translation initiation by linear scanning and internal ribosome binding

• Main Author: Ali, I. K.
• Published: University of Cambridge 2002
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595445

The experiments described in this dissertation used the rabbit reticulocyte lysate translation assay system to investigate what parts of the eIF4F complex are needed for (a) initiation by the scanning ribosome mechanism, and (b) IRES-dependent initiation. To this end, an affinity chromatography method for depleting reticulocyte lysates of eIF4G was developed, in which translation of all mRNAs was compromised, except (as predicted) translation driven by the HCV and pestivirus IRESes. The activity of the FMDV IRES, and that of the closely related encephalmoyocarditis virus (EMCV), in the depleted system was efficiently rescued by low concentrations of the p100 fragment of eIF4G, or even by just the central domain (p50). Deletions from the N-terminus of p50 did not seriously reduce this activity until they invaded the eIF4A interaction site. Surprisingly, a fragment consisting of amino acids 697-969 of eIF4GI was active in supporting IRES-dependent initiation even though it appears to lack the site of interaction with eIF3. Contrary to all expectations, the translation of capped mRNAs by the scanning ribosome mechanism was also restored by these eIF4G fragments which lack an eIF4E binding site, although high concentrations of p100 were needed than for the rescue of EMCV IRES activity. p100 or p50 could also support capped mRNA translation in normal reticulocyte lysates in which the function of the eIF4F complex had been impaired by addition of either m⁷GpppG cap analogue, or 4E-binding protein-1 (which removes eIF4E from the eIF4F complex), or FMDV L-greatly influenced by the precise nature of the 5’-terminus, whether uncapped or capped with m⁷GpppG, GpppG or ApppG end groups. Deletion of just 54 amino acids from the N-terminus of p50 resulted in a severe loss of activity towards scanning-dependent initiation, but had little; effect on the function of the EMCV IRES.