Dokument

Summary:
Identification of rnpB and rnpA in the Aquificales. Since the Aquificales (the close relatives of A. aeolicus) represent a group of bacteria for which not much information about RNase P RNA is available, we wanted to scrutinize if other members of this subphylum beside A. aeolicus also display idiosyncrasies regarding this essential enzyme. In cooperation with a bioinformatic research group (Gerhard Steger, Düsseldorf) we identified the P RNA (rnpB) and protein (rnpA) genes in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. Their P RNAs adhere to the bacterial P RNA consensus and proved to be active catalysts at high metal ion concentrations in vitro and could be activated by heterologous bacterial P proteins at low salt. Both RNAs lack helix P18 and thus one of the three tetraloop-helix interactions that bridge S-and C-domains. S. azorense and P. marina P RNA are more thermostable than E. coli P RNA and require higher temperatures for proper folding. The P protein genes (rnpA) of S. azorense and P. marina were identified as well and co-localize with the rmpH gene encoding ribosomal protein L34 as in the majority of bacteria. We also identified RNase P activity in other Aquificales (Aquifex pyrophilus, Hydrogenobacter thermophilus TK 6 and Thermocrinis ruber) and demonstrated that active RNase P holoenzymes can be reconstituted from their total RNA upon addition of the E. coli or B.subtilis P protein. We were further able to demonstrate that a close relative of A. aeolicus, A. pyrophilus, is also lacks the rnpA gene in the canonical bacterial genomic context. Finally, we succeeded in detecting RNase P activity in fractions of A. aeolicus cell lysates and demonstrated that the enzyme possesses an essential protein component that, unlike in other bacterial RNase P enzymes, cannot be substituted for by E. coli or B. subtilis P proteins. Strucural basis of a ribozyme’s thermostability: P1-L9 interdomain interaction in RNase P RNA. The two independent folding domains of type A bacterial P RNAs are interconnected by three long-range tertiary interaction: P1-L9, L8-P4 and P8-L18. Though predicted by phylogenetic analyses and confirmed by X-ray crystallography, the precise structural and functional role of these interactions is largely unclear. Our analysis of the P RNAs from the thermophilic Aqificales S. azorense and P. marina (see above) revealed that these P RNAs and the one from the thermophile Thermus thermophilus share a 5'-GYAA L9 tetraloop and a P1 receptor site consisting of a G-C bp tandem, a combination not present in other bacteria. Also, helices P1 and P9 were observed to be stabilized in P RNAs from thermophiles by helix extension and/or deletion of nucleotide bulges. These observations prompted us to scrutinize the importance of the P1-L9 interaction in P RNA of the thermophile Thermus thermophilus and to compare it to that of the mesophile E. coli. The P1-L9 contact indeed turned out to be crucial for folding and activity of T. thermophilus P RNA at high temperatures and low magnesium ion concentrations as present in holoenzyme reactions. I showed by native PAGE that the P1-L9 interaction module represents the key anchoring points towards folding into the most active RNA conformer of T. thermophilus P RNA. In contrast, disruption of this interaction in the P RNA from the mesophile E. coli did not abrogate functionality in vivo or in vitro. However, exchanging the P1-L9 module in E. coli P RNA for that of T. thermophilus generated a thermostable E. coli variant. I also replaced the P1-L9 interaction module in E. coli P RNA with an alternative pseudoknot interaction module present in some Mycoplasma P RNAs, which again resulted in thermostabilization of the chimeric P RNA. This work for the first time demonstrates that the module P1-L9 is directly linked to thermostabilization of P RNAs. This finding is an important step towards understanding structure-function relationships of catalytic RNAs.

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