The Yeast L30 Kink Turn RNA‐ Protein Interaction

Abstract

The L30e protein in Saccharomyccs cerevisiae is an autoregulatory protein that binds to its RNA transcript to inhibit splicing and to its mRNA to repress its translation. In both cases the RNA folds into a helix-internal loop-helix secondary structure with an asymmetric 2+5 purine-rich loop. The structure of the protein-RNA complex has recently been solved, and the L30 RNA has the structure of a kink-turn or K-turn RNA. We report the results of a series of in vitro and in vivo mutation experiments designed to compare the L30 RNA to other K-turn RNAs and to explore amino acid substitutions at the RNA-binding interface. An E. coli based two plasmid system was developed to measure in vivo repression ratios that correlated with RNA-protein binding affinities. The K-turn RNA motif is comprised of a canonical stem composed of Watson-Crick pairs, three unpaired nucleotides, and a non-canonical stem having two G:A pairs. Our results show that mutation of any of these Gs or As or reversing the pair dramatically weakens protein binding. In contrast, mutation of the three unpaired nucleotides was somewhat better tolerated. RNA mutants whose binding to the L30e protein was weakened were then used to find protein suppressors. Structural work shows that G56 is unpaired and stacked between the RNA A12 and Phe 85 of the protein. Mutation to U weakens protein binding to wild type protein but replacement of Gly 88 to Ser specifically restores binding to the mutant RNA. Gly88Ala and Gly88Cys suppress the G56U mutation to a lesser extent. A key determinant for strong RNA binding is Phe 85 that stacks on G56 and caps off one helix. Work in our laboratory shows that Phe85Ala greatly weakens binding, but that substitution by Trp, Tyr, His, and Ile are all tolerated. Interestingly, Trp and Ile are never found in L30 phylogenies but Ile is found in numerous proteins that bind K-turn RNAs.