A determination of the identity elements in yeast 18 S ribosomal RNA for the recognition of ribosomal protein YS11: the role of the kink-turn motif in helix 11.
A description of the site of interaction of YS11, the yeast homolog of eubacterial S17, with 18 S rRNA was obtained by assessing the binding of the ribosomal protein, in a filter retention assay, to oligoribonucleotides that reproduce regions of 18 S rRNA. YS11 binds predominantly to domain I; the Kd value is 113 nM. The dimensions of the YS11 binding site were refined, guided by chemical protection data and by the atomic structure of the Thermus thermophilus 30 S subunit, which has the S17 recognition site in 16 S rRNA. An oligoribonucleotide that mimics helix 11, a phylogenetically conserved region in domain I, binds YS11 with a Kd value of 230 nM; a second oligoribonucleotide that contains only the kink-turn motif in helix 11 binds YS11 with a Kd value of 528 nM. Thus, helix 11 has most of the nucleotides required for the recognition of YS11. To identify those nucleotides a set of 27 transversion mutations in H11 was constructed and their contribution to the binding of YS11 determined. Mutations of nine nucleotides (U313, C314, A316, G337, C338, G347, U348, U350, and C351) increased the Kd value for YS11 binding by at least eightfold; G325U and U349A mutations increased the Kd value fivefold. Eight of the 11 mutations are in the kink-turn in H11, confirming the critical importance of the motif for YS11 recognition. The other three nucleotides are in the lower stem and the terminal loop of H11, which makes a lesser, but still important, contribution to YS11 binding. The identity elements for YS11 recognition are: A316, G325, G337, G347, U348, U349, U350, and C351. The effect of the other nucleotides that decrease binding is probably indirect, presumably they affect the conformation of the binding site but do not have contacts to YS11 amino acid residues. The eight identity element nucleotides are in regions of H11 that deviate from A-form geometry and the contacts are predominantly, if not exclusively, to backbone phosphate and sugar oxygen atoms, indicating that YS11 recognizes the shape of the rRNA binding site rather than reading the sequence of nucleotides.
Dresios
John
J
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Chan
Yuen-Ling
YL
Wool
Ira G
IG
D000595:Amino Acid Sequence; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D012337:RNA, Ribosomal, 18S; D012269:Ribosomal Proteins; C045454:ribosomal protein S11