Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript

Abstract

Yeast ribosomal protein L30 binds to an asymmetric, purine-rich internal loop in its transcript to repress its own splicing and translation. The protein-bound form of the stem-internal loop–stem RNA is an example of a kink-turn RNA structural motif. Analysis of kink-turn motifs reveals that in (2 + 5) internal loops, the identities of five nucleotides are very important, while the remaining two may be varied. Previous SELEX experiments on the L30 binding site showed an identical pattern of sequence variation with five nucleotides highly conserved and two positions variable. In this work, internal loop residues were mutated and tested for protein binding in vitro and in vivo. The two sheared G-A pairs, which cannot be mutated without severely weakening L30 binding, make sequence specific contacts with other portions of the RNA and L30 protein. In contrast, the lone nucleotide that protrudes into the protein and an unpaired adenosine make no sequence-specific contacts, and may be mutated without compromising L30 binding. The internal loop allows the formation of a very tight bend that brings the two stems together with cross-strand stacking of two adenines and an interhelical ribose contact. Replacement of a ribonucleotide with a deoxynucleotide adjacent to the internal loop weakens protein binding significantly. In the absence of L30, some of the internal loop residues involved in the formation of the kink-turn motif are protected from chemical modification, indicating that some elements of kink-turn structure may form in the free L30 RNA.

Keywords

• RNA-binding protein
• RNA internal loop
• yeast ribosomal protein
• chemical modification
• kink-turn motif
• CMCT, 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate
• DMS, dimethylsulfate
• BSA, bovine serum albumin
• TBDMS, t-butyldimethylsilyl
• NOE, Nuclear Overhauser Effect
• SELEX, systematic evolution of ligands by exponential enrichment
• IPTG, isopropylthiogalactose