Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T. kodakaraensis transformants could be selected by ⌬trpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was ϳ8-fold higher than chromosome expression. An idealized ribosome binding sequence (5-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA-plus six-histidinetagged RpoL subunit (RpoL-HA-his 6 ) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni 2؉ binding and imidazole elution.Archaea, in common with Bacteria, have small circular genomes not encased in a nuclear compartment, with many genes organized and cotranscribed in operons. Archaeal genome replication and expression machineries, however, have many features more similar to their eukaryotic than to their bacterial counterparts (1,5,9,15). Progress in understanding archaeal replication and gene expression has been made using purified components in vitro, but in vivo validation of the results so obtained has been limited by the lack of genetic systems. This shortcoming has been most pronounced for the thermophilic and hyperthermophilic Euryarchaea, many of which are the foci of physiological and biochemical investigations (5, 9). Fortunately progress is now being made, most notably with Thermococcus kodakaraensis (2, 8), since it was discovered that T. kodakaraensis is naturally competent for DNA uptake and incorporates donor DNA into its genome by homologous recombination (25, 27). Deletion and mutation of chromosomal genes have resulted in the identification of novel biochemical pathways and facilitated the dissection of several events in archaeal transcription in T. kodakaraensis (12-14, 17, 19, 22, 23, 26, 28). To overcome the need for homologous recombination, we have now constructed shuttle vectors that replicate and express genes in both T. kodakaraensis and Escherichia coli. By using plasmid expression, we have documented and quantified the roles of a ribosome binding sequence (RBS) and alternative initiation codons in archaeal translation in T.kodakaraensis. We have also established that if one subunit (RpoL) of the multisubunit archaeal DNA-dependent RNA polyme...
Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His6-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His6-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.
SUMMARYArchaeal RNA polymerases (RNAPs) are most similar to eukaryotic RNAP II (Pol II) but require the support of only two archaeal general transcription factors, TBP (TATA-box binding protein) and TFB (archaeal homologue of the eukaryotic general transcription factors TFIIB) to initiate basal transcription. However, many archaeal genomes encode more than one TFB and/or TBP leading to the hypothesis that different TFB/TBP combinations may be employed to direct initiation from different promoters in Archaea. As a first test of this hypothesis, we have determined the ability of RNAP purified from Thermococcus kodakaraensis (T.k.) to initiate transcription from a variety of T.k. promoters in vitro when provided with T.k. TBP and either TFB1 or TFB2, the two TFBs encoded in the T.k. genome. With every promoter active in vitro, transcription initiation occurred with either TFB1 or TFB2 although the optimum salt concentration for initiation was generally higher for TFB2 (~250 mM K + ) than for TFB1 (~200 mM K + ). Consistent with this functional redundancy in vitro, T.k. strains have been constructed with the TFB1-(tfb1; TK1280) or TFB2-(tfb2; TK2287) encoding gene deleted. These mutants exhibit no detectable growth defects under laboratory conditions. Domain swapping between TFB1 and TFB2 has identified a central region that contributes to the salt sensitivity of TFB activity, and deleting residues predicted to form the tip of the B-finger region of TFB2 had no detectable effects on promoter recognition or transcription initiation but did eliminate the production of very short (≤ 5 nt) abortive transcripts.
Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a -glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5-TTTTTTTT (T 8 ) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t mcrA ) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression ϳ90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region. Template DNAs were amplified from each T. kodakarensis strain, and transcription in vitro by T. kodakarensis RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5-AAAAAAAA (A 8 ) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination.Archaea are prokaryotes, with only one RNA polymerase (RNAP), genes cotranscribed in operons, and translation and transcription coupled (5, 10, 12). Archaeal RNAPs, however, are more similar to the three eukaryotic RNAPs (polymerases [Pol] I, II, and III) than to bacterial RNAPs (9,19,24,25,29,48). In the absence of genetics, virtually all studies of archaeal transcription have used purified proteins and in vitro assays, and almost all have focused on initiation (13,15,29,33,36). Consistent with the Pol II-like structure of archaeal RNAPs, these studies have established that archaeal promoters have TATA boxes and transcription factor B recognition elements (BRE) and that initiation requires homologues of the eukaryotic TATA box binding protein (TBP) and transcription factor IIB (designated TFB in Archaea). Initiation is regulated by sequence-specific transcription factors that prevent or stimulate TBP, TFB, and/or RNAP binding to the promoter region (3, 13).In contrast to initiation, and despite the substantial differences between bacterial and eukaryotic transcription termination (4,6,8,14,16,21,27,30,(49)(50)(51), very little research has addressed transcription termination in Archaea. The few in vitro studies reported (36,41,43) are consistent in concluding that termination is stimulated by oligo(T) sequences, but the extents of termination observed differed substantially, and identical oligo(T)-containing sequences positioned at different sites on the same template elicited different termination responses. Unlike intrins...
Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second -glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ϳ7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of -glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.
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