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The La protein is a ubiquitous nuclear phosphoprotein that recognizes the 3' uridylates found in all newly synthesized RNA polymerase III transcripts. La binding stabilizes these transcripts from exonucleases and may also assist their folding. Here we present the first structural insights into how the La protein specifically interacts with its RNA substrates. The most conserved region of the La protein is the La motif, a domain also found in several other RNA-binding proteins. We have determined the structure of the La motif from the Trypanosoma brucei La protein to 1.6 A resolution (PDB code 1S29). The La motif adopts a winged helix-turn-helix architecture that has a highly conserved patch of mainly aromatic surface residues. Mutagenesis experiments support a critical role for this patch in RNA binding and show that it partly determines binding specificity for RNAs ending in 3' hydroxyl, a defining characteristic of the La protein. These findings reveal that the La motif is essential for high-affinity binding and also contributes to specificity.

The La protein protects the 3' ends of many nascent small RNAs from exonucleases. Here we report that La is required for efficient folding of certain pre-tRNAs. A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth. When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated. The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro. Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo. Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem. Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature. These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo.