Detection of a key tertiary interaction in the highly conserved GTPase center of large subunit ribosomal RNA.
Searches of ribosomal RNA sequences for compensatory base changes preserving Watson-Crick base pairing have led to detailed models of the conserved secondary structures of these RNAs. In principle, tertiary interactions can also be detected by searches for phylogenetically covariant bases. Within a highly conserved region of the large subunit ribosomal RNA termed the "GTPase center," the bases G-1056-U-1082.A-1086 are found in all eubacteria (Escherichia coli numbering), while A-1056.C-1082.G-1086 are found at the homologous positions in eukaryotes; archaebacteria fall into either category with some exceptions. Either sequence can potentially form a similar set of hydrogen bonds connecting the 3 bases. To determine the contribution of these 3 bases to RNA tertiary structure, sequence variants were made in RNA fragments covering the GTPase center. Correct folding of the RNA fragments was assayed by measuring the binding affinities of two different ligands that recognize the RNA tertiary structure: the highly conserved ribosomal protein L11, which is normally associated with the GTPase center RNA, and the peptide antibiotic thiostrepton, which inhibits the GTPase activity of eubacterial and some archaebacterial ribosomes. The results strongly support the existence of a base pair between positions 1082 and 1086: single mutations at either position weaken both L11 and thiostrepton binding by approximately 10-fold or more, while compensatory double mutations bind the ligands nearly as well as the wild-type E. coli sequence. Variants at position 1056 have little effect on either L11 or thiostrepton binding; a 3-base interaction is therefore not supported by these experiments. A base pair between positions 1082 and 1086 strongly constrains the geometry with which three helical segments join in the middle of the GTPase center.
Ryan
P C
PC
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
Draper
D E
DE
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics*; D014644:Genetic Variation; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics*; D013883:Thiostrepton / Q000378:metabolism
D012338:RNA, Ribosomal, 23S; D020558:GTP Phosphohydrolases; D013883:Thiostrepton