Recognition determinants for proteins and antibiotics within 23S rRNA.
Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecular genetic and biochemical probing techniques, we have concentrated on regions of the rRNA that are connected with specific functions. These are located in different domains within the 23S rRNA and include the ribosomal GTPase-associated center in domain II, which contains the binding sites for r-proteins L10.(L12)4 and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations here and by modification of A2058 by ErmE methyltransferase. ErmE recognizes a conserved motif displayed in the primary and secondary structure of the peptidyl transferase loop. Within domain VI of rRNA, the alpha-sarcin stem-loop is associated with elongation factor binding and is the target site for ribotoxins including the N-glycosidase ribosome-inactivating proteins ricin and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains are constrained by tetiary interactions, including a pseudoknot in domain II and long-range base pairings in the center of the molecule that bring domains II and V closer together. The phenotypic effects of mutations in these regions have been investigated by expressing 23S rRNA from plasmids. Allele-specific priming sites have been introduced close to these structures in the rRNA to enable us to study the molecular events there.
Douthwalte
S
S
Department of Molecular Biology, Odense University, Denmark.
Voldborg
B
B
Hansen
L H
LH
Rosendahl
G
G
Vester
B
B
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins