Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns.
The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition.
Allain
F H
FH
MRC Laboratory of Molecular Biology, Cambridge, UK.
Varani
G
G
D001482:Base Composition; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D007438:Introns / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D014498:Uracil / Q000737:chemistry
D002413:Cations, Divalent; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D008345:Manganese; D014498:Uracil; D006147:Guanine