Chemical crosslinking of ribonuclease P holoenzyme from Escherichia coli: Structure and catalytic mechanism of a ribozyme
Description
The structure of the Escherichia coli ribonuclease P (RNase P) holoenzyme was investigated by site-directed attachment of an aryl azide crosslink reagent to specific sites in the protein subunit of the enzyme. The protein subunit's interaction with a self-cleaving RNase P RNA-mini precursor-tRNA chimera (TPT118 RNA) was also investigated. The sites of crosslinking were mapped by primer extension to several conserved residues and structural features throughout the RNA. Probing the interaction between the protein subunit and TPT118 RNA allowed the confirmation of activity of crosslink sites observed with the RNase P RNA. The results of crosslinking to RNase P RNA suggest rearrangement of current tertiary models of the RNA subunit, particularly in regions poorly constrained by earlier data. Crosslinks from the protein subunit to the substrate precursor-tRNA (ptRNA) were also detected, consistent with previous crosslinking results in the Bacillus subtilis RNase P holoenzyme The interaction of the 5' end of ptRNAs with varying 5' leader lengths with the RNA subunit and holoenzyme was determined using an aryl azide crosslink reagent attached to the 5' end of the ptRNAs. The sites of crosslinking on the RNA subunit were mapped by primer extension. The sites of crosslinking to the P RNA were in agreement with previous results showing regions of the P RNA which interact with the 5' leader of ptRNA close to site of cleavage in both the B. subtilis and E. coli systems. The protein subunit blocks crosslinking sites on the P RNA in the holoenzyme when the 5' leader of ptRNAs is ≥3 bases long. These results are comparable to previous studies showing that the protein subunit interacts with the 5' leader of the substrate ptRNA and places the protein subunit next to the active site of RNase P RNA How the protein subunit affects the RNA subunit's preference in binding substrate as compared to product was analyzed using inhibition assays under single-turnover conditions. The protein subunit confers a 20-fold enhancement in the RNA subunit's binding of substrate to product. The RNA subunit alone had no preference in the binding of substrate and product. These results are in agreement with prior studies showing that the protein subunit increases the binding affinity of substrate as compared to product by the RNA subunit. Also, the importance of the universally conserved 3' CCA tail of tRNAs for binding was tested in the inhibition assays. The results confirmed previous reports that the 3' CCA is important in the RNA alone reaction, but is dispensable in the holoenzyme under single-turnover conditions