For EMSA, 6xHis-Flag tagged Tef4p was purified from a yeast tef4D strain with anti-FLAG M2-agarose affinity resin. Different concentrations (0.6, 0.5 and 0.4 pmol) of HF-Tef4p protein was used for incubation with 0.2 pmol of 32 P-labeled SL3/2/1(+) RNA or mutated RNAs at 25uC in a binding buffer [50 mM Tris-HCl (pH 8.2), 10 mM MgCl 2 , 10 mM DTT, 10% glycerol, 2 U of RNase inhibitor (Ambion)]. Samples were incubated at 25uC for 15 min, then resolved in 4% nondenaturing polyacrylamide gel [23] . Similar experiments were also performed with 6xHis-affinity purified recombinant Tef3p obtained from E. coli (not shown).