The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo.
The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3'-CCA termini (G292-C(75) and G293-C(74)). Here, we analyzed the phenotypes associated with disruption of the G292-C(75) or G293-C(74) pair in vivo. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW, in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G(74) base pair, but not a C292-G(75) pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg(2+) concentrations (2 mM), which we infer to be on the level of catalytically important Mg(2+) recruitment. At 4.5 mM Mg(2+), activity of mutant relative to the wild-type holoenzyme, was decreased only about twofold, but 13- to 24-fold at 2 mM Mg(2+). Moreover, our findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity, or RNA degradation. However, native PAGE experiments revealed nonidentical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, we cannot exclude that altered folding of the mutant RNAs may have also contributed to their in vivo defect.[1]References
- The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo. Wegscheid, B., Hartmann, R.K. RNA (2006) [Pubmed]
Annotations and hyperlinks in this abstract are from individual authors of WikiGenes or automatically generated by the WikiGenes Data Mining Engine. The abstract is from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.About WikiGenesOpen Access LicencePrivacy PolicyTerms of Useapsburg
