UBC Theses and Dissertations
An archaebacterial ribosomal protein gene cluster Shimmin, Lawrence Charles
The eubacteria, archaebacteria and eucaryota evolved from a common ancestral state, the progenote, approximately 4,000 million years ago. The archaebacteria flourish in extreme environments, exhibiting unusual macromolecular structures and metabolism of which much has recently been elucidated. Less, however, is known of the genetics of archaebacteria. In order to investigate gene structure, organization, regulation and evolution in the archaebacteria a gene cluster encoding the ribosomal proteins of the GTPase domain was cloned from the extremely halophilic archaebacterium Halobacterium cutirubrum, characterized and compared with the homologous genes and proteins from eubacteria and eucaryota. A clone containing a 5146 basepair insert of genomic Halobacterium cutirubrum NRCC 34001 DNA encoding the GTPase domain ribosomal proteins was characterized and discovered to retain the identical gene order (i.e. L11e, Lie, L10e and L12e) as the homologous Escherichia coli genes and in addition two transcribed upstream open reading frames encoding the potential proteins ORF, of unknown function and NAB, bearing sequence similarity to nucleic acid binding proteins. The predominant transcripts are monocistronic L11e and tricistronic Lie - L10e - L12e transcripts; monocistronic NAB and bicistronic NAB - L11e transcripts are present at reduced levels and the ORF is present as a very rare transcript. Common elements upstream of the transcription initiation sites include the motif TTCGA ... 4-15 bp ... TTAA ... 20-26 bp ... A or G transcription start. The NAB and some of the ORF transcripts are divergently transcribed from a single TTAA promotor element. The NAB and some of the ORF transcripts initiate 1 nucleotide before the coding region; the L11e monocistronic transcript initiates precisely at the first A of the initiator methionine ATG codon. The Lie - L10e - L12e tricistronic transcript has a 75 nucleotide leader that is probably involved in the autogenous regulation of the transcript at the translational level by the Lie protein. Termination of transcription occurs, with a single exception, within T tracts after GC rich regions. Although classic Shine-Dalgarno (eubacterial) type ribosome binding sites are present upstream of the Lie and L10e genes, the mechanism of translation initiation for transcripts with nil or negligible 5' leaders remains to be elucidated. Alignments between the deduced amino acid sequences of the L1le, Lie, Ll0e and L12eribosomal proteins and other available homologous proteins of archaebacteria, eubacteria and eucaryota have been made and show that the L11e, Lie and L10e proteins are colinear whereas the L12e protein has suffered a rearrangement through what appears to be gene fusion events. The L11e proteins exhibit (i) sequence conservation in the region interacting with release factor 1, (ii) conserved proline residues (probably contributing to the elongated shape of the molecule) and (iii) sites of methylation in Eco L11 are not conserved in the archaebacterial L11e proteins. The Lie proteins have regions of very high sequence similarity near the center and carboxy termini of the proteins but the relationships between protein structure and function remain unknown. Intraspecies comparisons between L10e and L12e sequences indicate the archaebacterial and eucaryotic L10e proteins contain a partial copy of the L12e protein fused to their carboxy terminus. In the eubacteria most of this fusion has been removed by a carboxy terminal deletion. Within the L12e derived region a 26 amino acid long internal modular sequence reiterated thrice in the archaebacterial L10e, twice in the eucaryotic L10e and once in the eubacterial L10e was discovered. This modular sequence also appears to be present in single copy in all Ll2e proteins and may play a role in L12e dimerization, L10e - L12e complex formation and the function of L10e - L12e complex in translation. From these sequence comparisons a model depicting the evolutionary progression gene cluster and proteins from the primordial state to the contemporary archaebacterial, eucaryotic and eubacterial states is presented.
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