Figure 4.

Mutating D27 reduces the specificity of La for RNAs ending in a 3′ hydroxyl. (A) La 1–192 (4.8 nM) was mixed with ³²P-labeled pre-tRNA_CCG^Arg (0.15 nM) in the presence of a 0- to 10 000-fold molar excess of unlabeled pre-tRNA_CCG^Arg terminating in either UUUp (left panel) or UUU_OH (right panel). Naked RNAs and RNPs were separated by native gel electrophoresis. The small differences in the fraction of RNA bound with 4.8 nM protein between this experiment and Figure 3C, lane 5, reflect experimental variation. Lane 1, no added protein. (B, C) The N23A mutant (B) or D27A mutant protein (C) was mixed with ³²P-labeled pre-tRNA_CCG^Arg (0.15 nM) in the presence of the indicated molar excess of unlabeled pre-tRNA_CCG^Arg as described in (A). The concentration of protein used in the reactions was 19.2 nM. Lane 1, no added protein. (D) Data from the competition titrations for La 1–192 and the D27A mutant are plotted as the fraction of labeled probe bound at each concentration of competitor RNA. Each data point represents the mean from at least three experiments. RNA concentrations are given in nM.