Determination of the Amino Acids in Yeast Ribosomal Protein YS11 Essential for the Recognition of Nucleotides in 18 S Ribosomal RNA

John Dresios,Yuen-Ling Chan,Ira G Wool

doi:10.1074/jbc.m513036200

Abstract

The nucleotides in domain I of 18 S rRNA that are important for the binding of the essential yeast ribosomal protein YS11 are mainly in a kink-turn motif and the terminal loop of helix 11 (H11). In the atomic structure of the Thermus thermophilus 30 S subunit, 16 amino acids in S17, the homolog of YS11, are within hydrogen bonding distance of nucleotides in 16 S rRNA. The homologous or analogous 16 amino acids in YS11 were replaced with alanine; nine of the substitutions slowed the growth of yeast cells. The most severe effects were caused by mutations R103A, N106A, K133A, T134A, and K151A. The T. thermophilus analogs of Arg103, Asn106, Thr134, and Lys151 contact nucleotides in the kink-turn motif of 16 S rRNA, whereas Lys133 contacts nucleotides in the terminal loop of H11. These contacts are predominantly with backbone phosphate and sugar oxygens in regions that deviate from A-form geometry, suggesting that YS11 recognizes the shape of its rRNA-binding site rather than reading the sequence of nucleotides. The effect of the mutations on the binding of YS11 to a domain I fragment of 18 S rRNA accorded, in general, with their effect on growth. Mutations of seven YS11 amino acids (Ser77, Met80, Arg88, Tyr97, Pro130, Ser132, and Arg136) whose homologs or analogs in S17 are within hydrogen bonding distance of nucleotides in 16 S rRNA did not affect binding. Apparently, proximities alone do not define either the amino acids or the nucleotides that are important for recognition.

Highlights

Otic ribosomal proteins [5,6,7,8,9], deriving most definitively from catalogs of the proximities of amino acids to nucleotides in the atomic structures of the Haloarcula marismortui 50 S [10] and Thermus thermophilus 30 S [11] ribosomal subunits
The primary binding site of YS11 is in domain I of 18 S rRNA, and the nucleotides important for recognition are located in the KT-11 motif and in the terminal loop of helix 11 (H11) [20]
Guided by the three-dimensional structure of TtS17 and 16 S rRNA in the T. thermophilus 30 S ribosomal subunit, we used reverse genetics and biochemical assays to determine the amino acids in YS11 that are important for the recognition of nucleotides in rRNA

Summary

EXPERIMENTAL PROCEDURES

Yeast and Bacterial Strains and Plasmids—Haploid S. cerevisiae strain LSF325 is a derivative of LSF327 in which the chromosomal ribosomal protein gene YS11-A was disrupted and replaced with a URA3 gene; haploid strain LSF326 is a derivative of LSF327 in which the chromosomal ribosomal protein gene YS11-B was disrupted and replaced with a LEU2 gene [22]. Plasmid pMALc2-YS11-B has the YS11-B open reading frame fused to the open reading frame of a maltose-binding protein (MBP) between the BamHI and SalI sites in the pMALc2 vector. This plasmid was used for the expression of recombinant wild-type and mutant YS11 proteins. A diploid strain produced by a cross of LSF325 and LSF326 was transformed with either wild-type or mutant pLFY204 plasmids, and the effect on yeast viability and growth was assessed following sporulation and tetrad analysis. Preparation of Plasmids Encoding YS11 and Expression and Purification of the MBP-YS11 Fusion Protein—A cDNA encoding ribosomal protein YS11 was generated by PCR from a yeast cDNA library; MBPYS11 was expressed in E. coli BL21(DE3) cells transformed with pMALc2-YS11-B and purified as described [20]. The Kd for the binding of mutant YS11 to RNA and the value relative to that for the binding to wild-type YS11 were determined in the same experiment

RESULTS AND DISCUSSION

Doubling time in YPD medium

Terminal amino acid deletions

Atomic contact Atoms in nucleotide