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. / Medicine, Faculty of / Medical Genetics, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/41498 |
| Date | January 1993 |
| Creators | Durovic, Peter Vincent |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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