A 2′-methyl or 2′-methylene group at G+1 in precursor tRNA interferes with Mg2+ binding at the enzyme-substrate interface in E-S complexes of E. coli RNase P
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Simona Cuzic
Abstract
We analyzed processing of precursor tRNAs carrying a single 2′-deoxy, 2′-OCH3, or locked nucleic acid (LNA) modification at G+1 by Escherichia coli RNase P RNA in the absence and presence of its protein cofactor. The extra methyl or methylene group caused a substrate binding defect, which was rescued at higher divalent metal ion (M2+) concentrations (more efficiently with Mn2+ than Mg2+), and had a minor effect on cleavage chemistry at saturating M2+ concentrations. The 2′-OCH3 and LNA modification at G+1 resulted in higher metal ion cooperativity for substrate binding to RNase P RNA without affecting cleavage site selection. This indicates disruption of an M2+ binding site in enzyme-substrate complexes, which is compensated for by occupation of alternative M2+ binding sites of lower affinity. The 2′-deoxy modification at G+1 caused at most a two-fold decrease in the cleavage rate; this mild defect relative to 2′-OCH3 and LNA at G+1 indicates that the defect caused by the latter two is steric in nature. We propose that the 2′-hydroxyl at G+1 in the substrate is in the immediate vicinity of the M2+ cluster at the phosphates of A67 to U69 in helix P4 of E. coli RNase P RNA.
©2007 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- 25 years of catalytic RNA: looking younger than ever!
- On the occasion of the 25th anniversary of the discovery of catalytic RNA
- An overview of the RNA world – for now
- Group II introns: structure, folding and splicing mechanism
- Expression of protein-coding genes embedded in ribosomal DNA
- Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs
- The spliceosome: a ribozyme at heart?
- A chemo-genetic approach for the study of nucleobase participation in nucleolytic ribozymes
- Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme
- A 2′-methyl or 2′-methylene group at G+1 in precursor tRNA interferes with Mg2+ binding at the enzyme-substrate interface in E-S complexes of E. coli RNase P
- Morphing the minimal and full-length hammerhead ribozymes: implications for the cleavage mechanism
- Idiosyncratic cleavage and ligation activity of individual hammerhead ribozymes and core sequence variants thereof
- RNA self-processing towards changed topology and sequence oligomerization
- Plasminogen-dependent internalization of soluble melanotransferrin involves the low-density lipoprotein receptor-related protein and annexin II
- Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?
