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Characterization of a novel archaeal RNA endonuclease from sulfolobus acidocaldarius Russell, Anthony George

Abstract

The study of the mechanisms and components involved in the complex ribosome biogenesis pathway has proven to be challenging, regardless of the organism chosen for analysis. While the strategies and catalytic components required to correctly fold, modify and cleave the pre ribosomal RNAs (rRNA) may differ between organisms from the archaeal, eubacterial or eukaryotic lineages; a unifying feature is the presence of spacer regions flanking the mature sequences which must be removed from the primary rRNA transcript during the maturation process. Some of the RNA nucleases that participate in precursor rRNA cleavages have been identified from several eubacterial and eukaryotic organisms, but those that generate the mature rRNA termini have, for the most part, remained elusive. In the Archaea, little is known about any of the components that are involved in pre rRNA cleavage and maturation. In this study, a novel archaeal RNA endonuclease from the hyperthermophile Sulfolobus acidocaldarius has been identified, characterized and partially purified. The analysis of the nuclease has been based upon its ability to cleave an in vitro synthesized RNA substrate containing the S.acidocaldarius 5' External Transcribed Spacer. The nuclease cleaves the substrate at (or near) positions that correspond to cleavage sites detected within in vivo rRNA precursors. The ability or inability of the enzyme to cleave mutagenized substrates has revealed some of its substrate recognition properties such as its absolute requirement for a purine nucleotide 5' to a cleavable phosphodiester bond. Interestingly, the essential RNA sequence elements recognized by the enzyme are found at the mature 5' end of the 16S rRNA, suggesting a possible in vivo role for the nuclease in mature 5' end formation. The nuclease activity has been partially purified and appears to have a size in excess of 600,000 Daltons. Attempts to obtain highly purified endonuclease activity have proven to be difficult due to the non-abundance, instability and unusual physical properties associated with the enzyme. While the protein constituents have not yet been identified, the activity does not appear to contain an essential RNA component, as was previously reported. The activity may associate with rRNA or ribosomal particles and its substrate requirements and physical characteristics suggest that this nuclease is indeed a novel archaeal endonuclease activity.

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