Figure 3.

Binding of wild-type and mutant La proteins to RNA. (A) ³²P-labeled yeast pre-tRNA_CCG^Arg (left panel) or pre-tRNA_CCG^Arg lacking the 9 nt 3′ trailer (right panel) was incubated without protein (lane 1) or with the truncated T. brucei La protein (LM+RRM; amino acids 1–192) at the indicated protein concentrations. The pre-tRNA concentration in the reaction was 0.15 nM. Naked RNAs and RNPs were separated in native gels. (B) ³²P-labeled yeast pre-tRNA_CCG^Arg (final concentration 0.15 nM) was incubated with either no protein (lane 1) or the indicated concentrations of full-length La (lanes 2–4), La 1–192 (lanes 5–8), the isolated LM (amino acids 1–89; lanes 9–11), the RRM (residues 94–192; lanes 12–14), or a 1:1 mixture of the LM and RRM (lanes 15–17). Naked RNAs and RNPs were separated by native gel electrophoresis. (C) Binding of La 1–192 (left panel) and the D27A mutant (right panel) to pre-tRNA_CCG^Arg was performed as described in (A). Protein concentrations in the reactions are indicated above the lanes. In lanes 1 and 10, no protein was added. We quantitated the difference in binding affinity of La 1–192 and the D27A mutant by performing multiple experiments. These revealed that La 1–192 bound the RNA with a K_d of 4.1±0.7 nM, while the D27A mutant bound the RNA with a K_d of 14.6±2.9 nM, an approximately 3.6-fold decrease in affinity. (D) pre-tRNA_CCG^Arg was subjected to gel shift analysis using either the wild-type La 1–192 (lanes 2–4) or the mutants Q14A/E16A (lanes 5–7), F29A (lanes 8–10), F50A (lanes 11–13), F17A (lanes 14–16), and Y18A (lanes 17–19). Protein concentrations in each lane are given in nM. Lanes 1 and 20, no protein.