RPL24B is required alongside eIF3h for efficient reinitiation after uORF translation. (A) Schematic of the uORF pattern in four constructs that differ in the length of the intercistronic spacer . uORFs 2a 2b and 3 each commence with a AUG codon in a strong Kozak context (asterisks) to minimize leaky scanning. Translational efficiency was estimated as previously described for Fig. 1. The spacer length dependence of reinitiation in the wild type is thought to reflect that acquisition of a fresh methionyl-tRNA by the 40S ribosome requires a minimal scanning time . Notably, rpl24b exhibits a similar reinitiation defect as eif3h. Bars denote standard error; n = 5-10; * P < 0.002 by Student's t-test. (B) The ATF4-like leader contains an additional uAUG in a strong context (A-3AAAUGG+4) that leads into an overlap-uORF. The new AUG lies 150nt downstream of the uORF 2/3 cluster and 60nt upstream of the FLUC AUG. Both rpl24b and eif3h mutants show poor FLUC expression in the presence or absence of the overlap uORF. Bars represent standard error (n = 8-12). (C) The similarity in translational activity between rpl24b and eif3h can also be seen on the 5' leader from yeast GCN4 when expressed heterologously in Arabidopsis (n = 5; bars denote standard error).