In vitro and in vivo investigations on the interaction of bacterial RNase P with tRNA 3’-CCA

The Ribonuclease P (RNase P) is a ribonucleoprotein enzyme, which catalyses the 5’-maturation of precursor tRNAs. Bacterial RNase P consists of one RNA subunit (P RNA; encoded by rnpB; ~400 nt), and a protein subunit (P protein, encoded by rnpA; ~120 aa). In vitro under elevated salt concentrations...

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Bibliographic Details
Main Author: Wegscheid, Barbara
Contributors: Hartmann, Roland K. (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2006
Pharmazeutische Chemie
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Summary:The Ribonuclease P (RNase P) is a ribonucleoprotein enzyme, which catalyses the 5’-maturation of precursor tRNAs. Bacterial RNase P consists of one RNA subunit (P RNA; encoded by rnpB; ~400 nt), and a protein subunit (P protein, encoded by rnpA; ~120 aa). In vitro under elevated salt concentrations the RNA subunit is catalytically active. However, under physiological conditions the protein subunit is essential for activity. Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences It could be demonstrated that structural type A and type B bacterial RNase P RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Even a single copy of E. coli rnpBwt integrated into the amyE site of the B. subtilis chromosome was sufficient for cell viability. The findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not critical for RNase P function in vivo, at least under standard growth conditions. 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-C75 and G293-C74). Here, the phenotyes associated with disruption of the G292-C75 or G293-C74 pair in vivo was analyzed. 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-G74 base pair, but not a C292-G75 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 Mg2+ concentrations (2 mM), which can be infered to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about 2-fold, but 13-24-fold at 2 mM Mg2+. Moreover, the 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 non-identical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, it cannot be excluded that altered folding of the mutant RNAs may have also contributed to their in vivo defect. In vivo role of bacterial type B RNase P interaction with tRNA 3’-CCA It has been unclear if catalysis by bacterial type B RNase P involves a specific interaction with p(recursor)tRNA 3’-CCA termini. We show that point mutations at two guanosines in loop L15 result in growth inhibition, which correlates with an enzyme defect at low Mg2+. For Bacillus subtilis RNase P, an isosteric C259-G74 bp fully and a C258-G75 bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base-pairing to tRNA 3’-CCA and emphasizing the importance of G258 identity. We infer the defect of the mutant enzymes to be primarily on the level of recruitment of catalytically relevant Mg2+, with a possible contribution from altered RNA folding. Cell viability of bacteria expressing mutant RNase P RNAs could be (partially) restored by RNase P protein overexpression, resulting in increased cellular RNase P levels. Finally, we demonstrate that B. subtilis RNase P is able to cleave CCA-less ptRNAs in vivo. We conclude that the in vivo phenotype upon disruption of the CCA interaction is either due to a global deceleration in ptRNA maturation kinetics or severe blockage of 5’-maturation for a subset of ptRNAs.
DOI:https://doi.org/10.17192/z2006.0907