UBC Theses and Dissertations
Characterisation of a novel pathway for ribosomal RNA maturation in Sulfolobus acidocaldarius Durovic, Peter Vincent
Since the initial proposition that the archaebacteria form a primary kingdom as distinct as that of the eubacteria or the eukaryotes, sequence data generated from the ribosomal RNA genes have flooded the databases and periodicals. Phylogenetic trees based on these sequences have been constructed to map the finest details of topology and branching order within the archaebacteria. Yet, despite the plethora of sequence data, relatively little was discovered regarding rRNAgene regulation, transcript processing and requirements for mature ribosome function. The aim of this study is to analyze possible novel regulatory mechanisms in the rRNA genes of the extremely thermoacidophilic archaebacterium Sulfolobus acidoccddccrius. The three ribosomal RNA genes were cloned and sequenced. The gene organization was confirmed to differ from that of the halophilic archaebacteria and the eubacteria: the 5S gene was not linked to the 16S and 23S operon, and the operon lacked recognizable tRNA sequences. Southern hybridization unveiled, and sequence data confirmed a long-standing confusion regarding species identity. The previously published Sulfolobus acidocaldarius 5S sequence was shown to have been attributed to the wrong species. Mapping experiments showed that both transcripts initiated downstream of a previously defined archaebacteria! promoter sequence. While sequence data showed the 5S transcript start site and end site to be coincidental with the mature 5S termini, the 16S-23S transcript was shown to contain a 143 nucleotide transcribed leader sequence, a 138 nucleotide intergenic sequence, and a trailer sequence of at least 105 nucleotides. Inverted repeat sequences within these transcribed non-coding regions allow for the formation of numerous stem-loops conforming to a semi-conserved archaebacterial structure. While no processing took place within the 5S transcript, extensive processing of the 16S-23S transcript was observed. Of the 12 processing sites mapped, only 6 could be accounted for in the context of precursor processing and maturation events known directly or inferred by analogy from the halophilic archaebacteria and the eubacteria. Alignment of the remaining sites revealed a non-trivial sequence and structural similarity. If the novel processing indeed took place in the postulated context, it would mark a radical departure from the expected maturation mechanism thought to predate the speciation of archaebacteria and eubacteria. To examine this possibility, in vitro transcripts from judiciously selected DNA fragments were subjected to cell-free extract. Analysis of the resultant cleavage products confirmed the presence not only of a novel processing activity mediated by a ribonucleoprotein complex but also of a novel processing pathway. Based on the locations of the novel processing sites within the primary 16S-23S transcript, a model for transcriptional regulation independent of polycistronic linkage is presented.