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John N Reeve

Publications and source records attributed to John N Reeve.

18 recordsLinked to original sources
PubMed2006

Archaeal minichromosome maintenance (MCM) helicase can unwind DNA bound by archaeal histones and transcription factors.

Protein-DNA complexes must be disassembled to facilitate DNA replication. Replication forks contain a helicase that unwinds the duplex DNA at the front of the fork. The minichromosome maintenance helicase from the archaeon Methanothermobacter thermautotrophicus required only ATP to unwind DNA bound into complexes by the M. thermautotrophicus archaeal histone HMtA2, transcription repressor TrpY, or into a transcription pre-initiation complex by M. thermautotrophicus TATA-box-binding protein, transcription factor B, and RNA polymerase. In contrast, the minichromosome maintenance helicase was unable to unwind DNA bound by this archaeal RNA polymerase in a stalled transcript-elongating complex.

Adenosine Triphosphate↗
PubMed2006

Archaeal histones and the origin of the histone fold.

Histone sequences have been identified in many archaeal genomes and in environmental samples, and they constitute a family of proteins that are structural homologs of the eukaryotic core histones. Most archaeal histones conform to the single histone-fold structural models that have been described, but a few histone variants exhibit short insertions, additional domains or fusions. Interpretation of these structural variations offers clues to the steps that might have occurred during the evolution and specialization of eukaryotic core histones.

Amino Acid Sequence↗
PubMed2005

Archaeal RNA polymerase is sensitive to intrinsic termination directed by transcribed and remote sequences.

Archaea are prokaryotes with a single DNA-dependent RNA polymerase (RNAP) that is homologous to, and likely resembles the ancestor of all three eukaryotic RNAPs. In vitro studies have confirmed that initiation by archaeal RNAPs resembles the Pol II system, and we report the first detailed in vitro investigation of archaeal transcription termination. Methanothermobacter thermautotrophicus (M.t.) RNAP is susceptible to intrinsic termination at an intergenic sequence that conforms to a bacterial intrinsic terminator, as well as at bona fide bacterial intrinsic terminators. In contrast to bacterial RNAPs, M.t. RNAP also terminated in response to synthetic and natural oligo-T-rich sequences that were not preceded by sequences with any recognizable potential to form a stable RNA hairpin. Both template topology and temperature influenced the position and extent of termination in vitro, and the results argue that transcription of an upstream sequence can alter the termination response of the archaeal RNAP at a remote downstream sequence.

Archaeal Proteins↗
PubMed2005

Archaeal chromatin proteins: different structures but common function?

Chromatin proteins promote chromosome flexibility in vivo, maintaining a compact yet decondensed template that permits polymerase accessibility. All Archaea have at least two types of chromatin proteins, and diversity in the chromatin protein population appears to prevent polymerization of a single type of protein. Of the numerous chromatin proteins that have been described in Archaea, only two--histones and Alba homologs--are present in all archaeal phyla. Although their structures and complexes with DNA have no similarities, their functions probably overlap as mutants that lack single chromatin proteins are viable.

Archaeal Proteins↗
PubMed2005

Use of a restriction enzyme-digested PCR product as substrate for helicase assays.

DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated.

Archaea↗
PubMed2005

Histones in crenarchaea.

Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.

Amino Acid Sequence↗
PubMed2005

Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus.

Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA.

Euryarchaeota↗
PubMed2004

Transcription by an archaeal RNA polymerase is slowed but not blocked by an archaeal nucleosome.

Archaeal RNA polymerases (RNAPs) are closely related to eukaryotic RNAPs, and in Euryarchaea, genomic DNA is wrapped and compacted by histones into archaeal nucleosomes. In eukaryotes, transcription of DNA bound into nucleosomes is facilitated by histone tail modifications and chromatin remodeling complexes, but archaeal histones do not have histone tails and archaeal genome sequences provide no evidence for archaeal homologs of eukaryotic chromatin remodeling complexes. We have therefore investigated the ability of an archaeal RNAP, purified from Methanothermobacter thermautotrophicus, to transcribe DNA bound into an archaeal nucleosome by HMtA2, an archaeal histone from M. thermautotrophicus. To do so, we constructed a template that allows transcript elongation to be separated from transcription initiation, on which archaeal nucleosome assembly is positioned downstream from the site of transcription initiation. At 58 degrees C, in the absence of an archaeal nucleosome, M. thermautotrophicus RNAP transcribed this template DNA at a rate of approximately 20 nucleotides per second. With an archaeal nucleosome present, transcript elongation was slowed but not blocked, with transcription pausing at sites before and within the archaeal nucleosome. With additional HMtA2 binding, complexes were obtained that also incorporated the upstream regulatory region. This inhibited transcription presumably by preventing archaeal TATA-box binding protein, general transcription factor TFB, and RNAP access and thus inhibiting transcription initiation.

Archaeal Proteins↗
PubMed2004

Transcription by Methanothermobacter thermautotrophicus RNA polymerase in vitro releases archaeal transcription factor B but not TATA-box binding protein from the template DNA.

Transcription initiation in Archaea requires the assembly of a preinitiation complex containing the TATA- box binding protein (TBP), transcription factor B (TFB), and RNA polymerase (RNAP). The results reported establish the fate of Methanothermobacter thermautotrophicus TBP and TFB following transcription initiation by M. thermautotrophicus RNAP in vitro. TFB is released after initiation, during extension of the transcript from 4 to 24 nucleotides, but TBP remains bound to the template DNA. Regulation of archaeal transcription initiation by a repressor competition with TBP for TATA-box region binding must accommodate this observation.

Archaeal Proteins↗
PubMed2003

Molecular identification of bacteria and Eukarya inhabiting an Antarctic cryoconite hole.

Inhabitants of a cryoconite hole formed in the Canada Glacier in the McMurdo Dry Valley region of Antarctica have been isolated and identified by small subunit (16S/18S) rDNA amplification, cloning, and sequencing. The sequences obtained revealed the presence of members of eight bacterial lineages (Acidobacterium, Actinobacteria, Cyanobacteria, Cytophagales, Gemmimonas, Planctomycetes, Proteobacteria, and Verrucomicrobia) and metazoan (nematode, tardigrade, and rotifer), truffle (Choiromyces), ciliate (Spathidium), and green algal (Pleurastrium) Eukarya. Bacterial recovery was approximately 20-fold higher at 4 degrees C and 15 degrees C than at 22 degrees C, and obligately psychrophilic bacteria were identified and isolated. Several of the rDNA molecules amplified from isolates and directly from cryoconite DNA preparations had sequences similar to rDNA molecules of species present in adjacent lake ice and microbial mat environments. This cryoconite hole community was therefore most likely seeded by particulates from these local environments. Cryoconite holes may serve as biological refuges that, on glacial melting, can repopulate the local environments.

Antarctic Regions↗
PubMed2003

Archaeal chromatin and transcription.

Archaea contain a variety of sequence-independent DNA binding proteins consistent with the evolution of several different, sometimes overlapping and exchangeable solutions to the problem of genome compaction. Some of these proteins undergo residue-specific post-translational lysine acetylation or methylation, hinting at analogues of the histone modifications that regulate eukaryotic chromatin structure and transcription. Archaeal transcription initiation most closely resembles the eukaryotic RNA polymerase II (RNAPII) system, but Archaea do not appear to have homologues of the multisubunit complexes that remodel eukaryotic chromatin and activate RNAPII initiation. In contrast, they have sequence-specific regulators that repress and perhaps activate archaeal transcription by mechanisms superficially similar to the bacterial paradigm of regulating promoter binding by RNAP. Repressors compete with archaeal TATA-box binding protein (TBP) and TFB for the TATA-box and TFB-recognition elements (BRE) of the archaeal promoter, or with archaeal RNAP for the site of transcription initiation. Transcript-specific regulation by repressors binding to sites of transcript initiation is consistent with such sites having very little sequence conservation. However, most Archaea have only one TBP and/or TFB that presumably must therefore bind to similar TATA-box and BRE sequences upstream of most genes. Repressors that function by competing with TBP and/or TFB binding must therefore also make additional contacts with transcript-specific regulatory sites adjacent or remote from the TATA-box/BRE region. The fate of the archaeal TBP and TFB following transcription initiation remains to be determined. Based on functional homology with their eukaryotic RNAPII-system counterparts, archaeal TBP and possibly also TFB should remain bound to the TATA-box/BRE region after transcription initiation. However, this seems unlikely as it might limit repressor competition at this site to only the first round of transcription initiation.

Amino Acid Sequence↗
PubMed2003

Bacterial recovery from ancient glacial ice.

Ice that forms the bottom 18 m of a 308 m ice core drilled from the Guliya ice cap on the Qinghan-Tibetan plateau in Western China is over 750000 years old and is the oldest glacial ice known to date. Fourteen bacterial isolates have been recovered from samples of this ice from approximately 296 m below the surface (mbs). Based on 16S rDNA sequences, these are members of the alpha- and beta-proteobacterial, actinobacterial and low-G + C Gram-positive bacterial lineages. 16S rDNA molecules have also been amplified directly, cloned and sequenced from the ice-core melt water. These originated from Pseudomonas and Acinetobacter gamma-proteobacterial species. These results demonstrate that bacteria can be recovered from water ice that has frozen for time periods relevant to biological survival through terrestrial ice ages or during interplanetary transport.

Bacteria↗
PubMed2003

Conserved eukaryotic histone-fold residues substituted into an archaeal histone increase DNA affinity but reduce complex flexibility.

Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.

Amino Acid Sequence↗
PubMed2002

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling.

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.

Amino Acid Sequence↗
PubMed2002

The genome of M. acetivorans reveals extensive metabolic and physiological diversity.

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic growth. Although motility has not been observed in any Methanosarcineae, a flagellin gene cluster and two complete chemotaxis gene clusters were identified. The availability of genetic methods, coupled with its physiological and metabolic diversity, makes M. acetivorans a powerful model organism for the study of archaeal biology. [Sequence, data, annotations and analyses are available at http://www-genome.wi.mit.edu/.]

Archaeal Proteins↗
PubMed2001

Both DNA and histone fold sequences contribute to archaeal nucleosome stability.

The roles and interdependence of DNA sequence and archaeal histone fold structure in determining archaeal nucleosome stability and positioning have been determined and quantitated. The presence of four tandem copies of TTTAAAGCCG in the polylinker region of pLITMUS28 resulted in a DNA molecule with increased affinity (DeltaDeltaG of approximately 700 cal mol(-1)) for the archaeal histone HMfB relative to the polylinker sequence, and the dominant, quantitative contribution of the helical repeats of the dinucleotide TA to this increased affinity has been established. The rotational and translational positioning of archaeal nucleosomes assembled on the (TTTAAAGCCG)(4) sequence and on DNA molecules selectively incorporated into archaeal nucleosomes by HMfB have been determined. Alternating A/T- and G/C-rich regions were located where the minor and major grooves, respectively, sequentially faced the archaeal nucleosome core, and identical positioning results were obtained using HMfA, a closely related archaeal histone also from Methanothermus fervidus. However, HMfA did not have similarly high affinities for the HMfB-selected DNA molecules, and domain-swap experiments have shown that this difference in affinity is determined by residue differences in the C-terminal region of alpha-helix 3 of the histone fold, a region that is not expected to directly interact with DNA. Rather this region is thought to participate in forming the histone dimer:dimer interface at the center of an archaeal nucleosome histone tetramer core. If differences in this interface do result in archaeal histone cores with different sequence preferences, then the assembly of alternative archaeal nucleosome tetramer cores could provide an unanticipated and novel structural mechanism to regulate gene expression.

Amino Acid Sequence↗