Subject(s)
Oligonucleotides/chemistry , RNA, Ribosomal/chemistry , Ribosomes/chemistry , Amines/chemistry , Amino Acids/chemistry , Chromatography, High Pressure Liquid , Cross-Linking Reagents/pharmacology , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Genetic Techniques , Models, Chemical , Models, Molecular , Nucleic Acid Conformation , Peptides/chemistry , RNA, Ribosomal/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 23S/chemistry , RNA-Directed DNA Polymerase/pharmacology , Ribonuclease H/pharmacology , Ribosomes/metabolism , Time Factors , Transcription, Genetic , Ultraviolet Rays , Water/metabolismABSTRACT
The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.
Subject(s)
Escherichia coli/enzymology , Peptidyl Transferases/chemistry , RNA, Ribosomal, 23S/chemistry , Ribosomes/enzymology , Adenine Nucleotides/chemistry , Base Sequence , Binding Sites , Molecular Sequence Data , Oligonucleotides/chemistry , Photolysis , RNA/chemistry , RNA, Bacterial/chemistry , Ribosomal Proteins/chemistry , Thionucleotides/chemistry , Uracil/chemistryABSTRACT
We report the synthesis of a radioactive, photolabile 2'-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called 'P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 A from 23S RNA nucleotides G2252 and G2253. PHONT1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.