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A Comparison between Information Processing in Archaea and Eukarya
Studying Information Processing
Living cells evolved complex systems to handle the flow of information both accurately and efficiently. These systems are highly comparable between the three domains of life: eukaryotes, bacteria and archaea. The central components of replication, transcription, aminoacylation, and translation are found in every living cell known today, with only relatively small deviations, despite a separation of billions of years of evolution. Archaea are unicellular, do not contain organelles, and have relatively small genomes, so are, at first sight, quite similar to their far better known prokaryotic cousins: the bacteria. Nevertheless, if it comes down to information processing, archaea are, surprisingly, more related to eukaryotes than to bacteria, both at the sequence level of RNA and proteins, and at the architecture level of key complexes as well. This makes them excellent model systems to study eukaryote-like information processing. The absence of cell specialization, less cell organization, less or even no intracellular compartmentalization, and less intensive regulation, have proven to give a clearer picture of the function of conserved key elements within these complex systems. [Chapter 2]
In this thesis, we report several attempts to elucidate functional details of some very conserved factors in information processing in S. solfataricus using recently established genetic modification techniques. S. solfataricus is a thermoacidophilic crenarchaeote that grows optimally at temperatures between 70°C and 85°C and at pH values between 2 and 3. Its genome sequence is known since 2001. Best practices have become standardized between laboratories, and the genomic toolbox includes gene knockout, overexpression systems, the availability of reporter genes, and tunable promoters.
MBF1, a highly conserved activator
MBF1 (multi-protein bridging factor 1) is reported to be a transcriptional co-activator in eukaryotes. It was shown to cross the gap between transcription regulators and the transcriptional machinery itself. MBF1 was found to be highly conserved within archaea, being present in almost all species with the key exception of marine thaumarchaeotes. However, none of the associated transcription regulators were known to be present within the archaeal domain, raising the question whether a class of other regulators was overlooked, or that archaeal MBF1 might be a transcriptional activator itself, binding to DNA directly instead of indirectly via a binding partner. Additionally one study revealed a surprising dual role of this protein: in yeast it was not only associated with transcription but contributed to translation fidelity as well. A neighbourhood analysis across the archaeal domain revealed no clear preference for either transcription or translation. Elements of both systems are equally present, especially in the well conserved neighbourhood within the crenarchaeotes. [Chapter 3]
A mbf1 disruption mutant of the S. solfataricus was made using heterozygous recombination with a suicide plasmid. Under standard laboratory growth conditions mbf1 appears to be not essential for growth, and comparing growth characteristics with its parental strain did not reveal striking differences between the two. It was observed, that the Sulfolobus mbf1 disruption mutant is much more sensitive during cultivation than its parental strain, showing sudden death during growth much more often. Being hard to quantify, this behaviour was especially observed when cultures were transferred at later stages during stationary phase or unfrozen from long term storage. But the largest difference was observed in the increased sensitivity of the mbf1 disruption mutant towards paromomycin. Paromomycin is an aminoglycoside-type antibiotic that interferes with the recognition of cognate codon-anti-codon binding within the ribosomes during translation. [Chapter 4]
A more detailed study to the molecular characteristics of the archaeal MBF1 from S. solfataricus revealed hardly any associations to the transcription machinery, but strengthened the assumed association to the translation apparatus. It was found that archaeal MBF1 consists of two domains that are structurally independent: an N-terminal zinc-ribbon, which is not conserved beyond the archaeal MBF1s, and the well conserved C-terminal HTH-domain (helix-turn-helix domain). This C-terminal HTH domain was shown to bind to the small ribosomal subunit by affinity purification, and in co-purification experiments, in which we detected the presence of archaeal MBF1 in ribosomal purifications. NMR structure comparisons confirmed that archaeal MBF1 binds to the small ribosomal subunit using its C-terminal HTH domain, whereas the N-terminal zinc-ribbon might only contribute to this interaction, but does not participate directly in binding. [Chapter 5]
Altogether, these findings made us believe that MBF1s in archaea are not associated with transcription but rather with translation. Based on the observations in yeast, and more recently its binding to polyadenylated mRNAs in different eukaryotic species, and, against the backdrop that the protein domain that binds to the small ribosomal subunit in S. solfataricus is highly conserved across the archaeao-eukaryotic lineage, it is tempting to speculate that the eukaryotic MBF1 plays a comparable role in the translation process in eukaryotes as well.
TGT, a conserved dichotomy
Another well conserved element within all three domains of life, which is involved in information processing, is the TGT (tRNA-guanine transglycosylase) family of proteins. This family of proteins shows a clear dichotomy: TGT is responsible for the exchange of guanine at the wobble position (position 34) of the anti-codon of certain tRNAs with either queuosine in eukaryotes or its precursor preQ1 in bacteria, whereas, in archaea, TGT is responsible for the exchange of guanine with preQ0 at position 15 in almost, if not all, archaeal tRNAs. PreQ0 is in a later stage converted to archaeosine by another protein that belongs to the TGT family as well.
Disruption of the tgt gene, which encodes the TGT protein in S. solfataricus, revealed that it was solely responsible for this process without any redundancy present. Like mbf1, this gene appeared to be non-essential, as this mutant was also as viable as its parental strain, and showed hardly any changes in growth characteristics. In comparison to the mbf1 disruption mutant, the tgt disruption mutant was much more stable and did not reveal the sensitivity to stationary phase. It grew slightly slower than the parental strain, especially at normal temperatures (75°C), but when temperature levels were raised (87-93°C) growth returned to almost wild-type levels. [Chapter 6]
Aiding research to the basal machinery of RNAP
Beyond doubt, the best studied, element of information processing systems is the RNAP (RNA polymerase) complex. Its basal core is present in all known life forms, and is highly conserved. The surrounding, auxiliary, and regulatory elements are less conserved, but, nevertheless, the archaeal RNAP is almost identical to the eukaryotic RNAP II complex (see figure). This high resemblance already proved beneficial, as the heterologous expression of the archaeal RNAP revealed numerous functional details about the molecular characteristics of the complex as a whole, and, in addition, revealed also an unprecedented insight in the separate subunits as this provided opportunities to tamper with the subunit composition and to modify the separate subunits themselves by introducing genetic variations.
Unfortunately, purification of homologously expressed complexes, which are expressed in archaeal systems itself, are, in contrast to ones heterologously expressed in bacterial hosts, hard to obtain, and involve a number of purification steps and therefore a substantial amount of biomass. To enable easier purification, a method was developed in which a purification tag was inserted in the genome of S. solfataricus after a gene that encodes an RNAP subunit, avoiding artificial overproduction by viral infections or heterologous expression in other less adapted hosts. In a proof of principle experiment, the enrichment an RNAP core component was proven, whereas an auxiliary element was tagged using this novel method. [Chapter 7]
|Qualification||Doctor of Philosophy|
|Award date||23 Feb 2015|
|Place of Publication||Wageningen|
|Publication status||Published - 2015|
- transfer rna
- rna polymerase
- regulation of transcription
- sulfolobus solfataricus