In all cell types, chromosomal DNA replication is a complex process entailing three enzymatic activities: helicase activity for double-helix unzipping and primase and DNA polymerase for RNA primer de novo synthesizing and elongation respectively [1,2].Based on the biochemical data accumulated to date, archaeal DNA replication involves a smaller number of polypeptides at each stage of the process and is thus just a simpler form of the much more complex eukaryotic replication machinery [3][4][5][6]. Nonetheless, Archaea are not simply 'mini Eukarya'. A better definition would be 'a mosaic of eukaryal and bacterial systems with specific archaeal features'. Aspects worth mentioning in this respect are the promiscuous nature of the nucleic acid functions performed by archaeal primases and the dual, template-dependent and A tri-functional monomeric primase-polymerase domain encoded by the plasmid pIT3 from Sulfolobus solfataricus strain IT3 was identified using a structural-functional approach. The N-terminal domain of the pIT3 replication protein encompassing residues 31-245 (i.e. Rep245) was modeled onto the crystallographic structure of the bifunctional primase-polymerase domain of the archaeal plasmid pRN1 and refined by molecular dynamics in solution. The Rep245 protein was purified following overexpression in Escherichia coli and its nucleic acid synthesis activity was characterized. The biochemical properties of the polymerase activity such as pH, temperature optima and divalent cation metal dependence were described. Rep245 was capable of utilizing both ribonucleotides and deoxyribonucleotides for de novo primer synthesis and it synthesized DNA products up to several kb in length in a template-dependent manner. Interestingly, the Rep245 primase-polymerase domain harbors also a terminal nucleotidyl transferase activity, being able to elongate the 3¢-end of synthetic oligonucleotides in a non-templated manner. Comparative sequence-structural analysis of the modeled Rep245 domain with other archaeal primase-polymerases revealed some distinctive features that could account for the multifaceted activities exhibited by this domain. To the best of our knowledge, Rep245 typifies the shortest functional domain from a crenarchaeal plasmid endowed with DNA and RNA synthesis and terminal transferase activity.
pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5 end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.Whereas major studies on viral systems in Bacteria and Eukarya have shed sufficient light on basic and regulated gene expression in these organisms, very little regarding the elucidation of the mechanisms of viral gene expression and regulation in hyperthermophilic Archaea has been addressed. A few archaeal viruses have been investigated in depth at molecular and physiological levels (23,25,48,53). These viruses are known to be highly unusual in terms of morphology and genome structure/sequence (38,39,40,41,45). A survey of the extrachromosomal elements in the hyperthermophilic crenarchaeon Sulfolobus (64, 65) has revealed the existence of many new viruses, with the Fuselloviridae being the most common family among the Sulfolobales to date (37). Sulfolobus spindleshaped virus 1 (SSV1) is the best-studied member of this family, and it behaves as a temperate virus both in Sulfolobus shibatae and in the nonnatural but related Sulfolobus solfataricus host (53); infection, integration of DNA into the host chromosome, and the production of virions cause apparently no phenotype change but do cause a significant growth retardation of the host cells, which can be visualized as turbid plaques on plated lawns of indicator host cells around propagation foci (53, 65).Transcription studies conducted on SSV1 have pointed out that the copy number of the episomal DNA as well as the virus titer remain essentially constant in the unirradiated host (48). DNA replication increases after induction by UV or other DNA-damaging agents and seems to be mediated by transcription at the promoter T ind (48,53). Furthermore, the structural genes of SSV1 are constitutively and coordinately transcribed in nonirradiated cells, and the amount of these...
The Escherichia coli/Sulfolobus solfataricus shuttle vector pEXSs was used as a cloning vehicle for the gene transfer and expression of two bacterial genes in Sulfolobus solfataricus. The alcohol dehydrogenase (adh) from the moderate thermophilic Bacillus stearothermophilus (strain LLDR) and a mutagenised version encoding a less thermostable ADH enzyme were the selected genes. S. solfataricus adh promoter and aspartate aminotransferase terminator were used to drive the heterologous gene expression and to guarantee the correct termination of the transcripts, respectively. The constructed vectors were found to be able to carry these 'passenger' genes without undergoing any rearrangements. The active transcription of bacillar mRNAs was ascertained in vivo by RT-PCR. Transformed S. solfataricus expressed functional exogenous ADHs that showed unaffected kinetic and chemical-physical features.
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