PMID:11160931
| Citation |
Hou, YM, Zhang, X, Holland, JA and Davis, DR (2001) An important 2'-OH group for an RNA-protein interaction. Nucleic Acids Res. 29:976-85 |
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| Abstract |
We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions. |
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| Keywords |
Acylation; Amino Acyl-tRNA Synthetases/chemistry; Amino Acyl-tRNA Synthetases/genetics; Amino Acyl-tRNA Synthetases/metabolism; Bacillus subtilis/genetics; Base Sequence; Cysteine/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen Bonding; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Molecular Structure; Nucleic Acid Conformation; Oligoribonucleotides/chemistry; Oligoribonucleotides/genetics; Oligoribonucleotides/metabolism; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Transfer, Cys/chemistry; RNA, Transfer, Cys/genetics; RNA, Transfer, Cys/metabolism; RNA-Binding Proteins/chemistry; RNA-Binding Proteins/genetics; RNA-Binding Proteins/metabolism; Uridine/analogs & derivatives; Uridine/chemistry; Uridine/metabolism |
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