UBC Theses and Dissertations
RNA guided nucleotide modification of ribosomal and non-ribosomal RNAs in archaea Ziesche, Sonia Madlen
Archaea use ribonucleoprotein (RNP) machines similar to those found in the eukaryotic nucleolus to methylate ribose residues in nascent ribosomal RNA. The archaeal complex required for this 2'-O-ribose-methylation consists of the C/D box sRNA guide and three proteins, the core RNA binding aL7a protein, the aNop56 protein and the methyltransferase fibrillarin protein. These RNP machines were reconstituted in vitro from purified recombinant components, and were shown to have methylation activity when provided with a simple target oligonucleotide, complementary to the sRNA guide sequence (Omer et al., 2002). The accuracy in directing methylation to the correct nucleotide in the target of the in vitro reconstituted C/D box RNP was shown. To obtain a better understanding of the versatility and specificity of this reaction, the activity of reconstituted particles on more complex target substrates (including 5S rRNA, tRNA[sup Gln] and "double guide" oligonucleotides that exhibit either direct or reverse complementarity to both the D' and D box guides) has been examined. The natural 5S rRNA and tRNA[sup Gln] substrates were efficiently methylated in vitro, providing that the complementarity between guide and target was approximately ten base pairs in length, and lacked mismatches. Maximal activity of double guide sRNAs required that both methylation sites be present in cis on the target RNA. These experiments defined the minimum number of components and conditions required to achieve in vitro, RNA guide directed 2'-O-ribose methylation of ribosomal and non-ribosomal target RNA.
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