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. Author manuscript; available in PMC: 2013 May 15.
Published in final edited form as: Nat Struct Mol Biol. 2012 Jul 1;19(8):744–753. doi: 10.1038/nsmb.2308

Figure 5.

Figure 5

Fun12 is responsible for GTP-stimulation of in vitro cleavage.

(a) Strain YSLD69 (PGAL::3HA-NOB1; PGAL::3HA-FUN12; pRS415-PTH-Nob1) was transformed with empty (empty) or PADH1::FUN12 plasmids. Following transfer to glucose medium for 12 h pre-40S particles were purified using PTH-Nob1 and cleavage at site D determined as in Fig. 2. (b) Cleavage efficiencies observed after addition of ATP or GTP were quantified relative to the control. The ratios of the activities observed from cell lysates expressing or depleted of Fun12 are presented (* P<0.2). (c) Site D cleavage in PTH-Nob1 pre-40S fractions isolated from strain YSLD69 carrying either a plasmid allowing low level expression of wild-type, non-tagged Fun12 under the control of a MET25 promoter or a plasmid expressing the Fun12D533N mutant that allows hydrolysis of XTP. Cells were transferred to medium containing 2% glucose and 8 mg l−1 methionine (2.5 fold lower than the normal concentration) for 10 h before pre-40S purification and analysis as in Fig. 2. (d) Activities induced by either GTP or XTP were quantified in comparison to the mock control. The ratio of the activities observed in cell lysates expressing Fun12 WT or mutant is presented (* P<0.2).