Structure of histone-based chromatin in Archaea.

Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Pseudomonas isolation and identification: an introduction to the challenges of polyphasic taxonomy.

The ability to isolate an organism in pure culture from the environment is a manageable task for undergraduate students; the identification of that organism requires integration of both genotypic and phenotypic data and illustrates the challenges inherent in contemporary bacterial taxonomy. In this ten-laboratory period series of exercises, students isolate a strain of Pseudomonas from soil and characterize its biochemical and physiological properties, as well as determine the DNA sequence of its 16S rRNA genes. Integrating these data positions students to defend their classification of the isolate as a new species or as a member of a validly described species. Assessment data demonstrate that both knowledge of and confidence in understanding of the principles of laboratory handling of Pseudomonas and bacterial taxonomy increased following the exercises.

Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer.

TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.9 Šresolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V(M)) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein.

Archaeal chromatin proteins histone HMtB and Alba have lost DNA-binding ability in laboratory strains of Methanothermobacter thermautotrophicus.

Alignments of the sequences of the all members of the archaeal histone and Alba1 families of chromatin proteins identified isoleucine residues, I19 in HMtB and I39 in MtAlba, in Methanothermobacter thermautotrophicus, at locations predicted to be directly involved in DNA binding. In all other HMfB family members, residue 19 is an arginine (R19), and either arginine or lysine is present in almost all other Alba1 family members at the structural site equivalent to I39 in MtAlba. Electrophoretic mobility shift assays revealed that recombinant HMtB and MtAlba do not bind DNA, but variants constructed with R19 and R39, respectively, bound DNA; and whereas MtAlba(I19) did not bind RNA, MtAlba(R19) bound both single stranded RNA and tRNA. Amplification and sequencing of MT0254 (encodes HMtB) and MT1483 (encodes MtAlba) from several Methanothermobacter thermautotrophicus lineages has revealed that HMtB and MtAlba had arginine residues at positions 19 and 39, respectively, in the original isolate and that spontaneous mutations must have occurred, and been fixed, in some laboratory lineages that now have HMtB(I19) and MtAlba(I39). The retention of these variants suggests some continuing functions and fusion of the HMtB(I19) sequence to HMtA2 resulted in a protein that folds to form a histone fold heterodimer that binds and compacts DNA. The loss of DNA binding by HMtB(I19) does not therefore prevent HMtB from participating in DNA interactions as one partner of an archaeal histone heterodimer.

TrpY regulation of trpB2 transcription in Methanothermobacter thermautotrophicus.

TrpY binds specifically to TRP box sequences upstream of trpB2, but the repression of trpB2 transcription requires additional TrpY assembly that is stimulated by but not dependent on the presence of tryptophan. Inhibitory complex formation is prevented by insertions within the regulatory region and by a G149R substitution in TrpY, even though TrpY(G149R) retains both TRP box DNA- and tryptophan-binding abilities.

Deletion of the archaeal histone in Methanosarcina mazei Gö1 results in reduced growth and genomic transcription.

HMm is the only archaeal histone in Methanosarcina mazei Göl and recombinant HMm, synthesized by expression of MM1825 in Escherichia coli, has been purified and confirmed to have the DNA binding and compaction properties characteristic of an archaeal histone. Insertion of a puromycin resistance conferring cassette (pac) into MM1825 was not lethal but resulted in mutants (M. mazei MM1825::pac) that have impaired ability to grow on methanol and trimethylamine. Loss of HMm also resulted in increased sensitivity to UV light and decreased transcript levels for approximately 25% of all M. mazei genes. For most genes, the transcript decrease was 3- to 10-fold, but transcripts of MM483 (small heat-shock protein), MM1688 (trimethylamine:corrinoid methyl transferase) and MM3195 (transcription regulator), were reduced 100-, 100- and 25-fold, respectively, in M. mazei MM1825::pac cells. Transcripts of only five adjacent genes that appear to constitute an aromatic amino acid biosynthetic operon were elevated in M. mazei MM1825::pac cells. Complementary synthesis of HMm from a plasmid transformed into M. mazei MM1825::pac restored wild-type growth and transcript levels.

Spontaneous trpY mutants and mutational analysis of the TrpY archaeal transcription regulator.

Over 90% of Methanothermobacter thermautotrophicus mutants isolated as spontaneously resistant to 5-methyl tryptophan had mutations in trpY. Most were single-base-pair substitutions that identified separate DNA- and tryptophan-binding regions in TrpY. In vivo and in vitro studies revealed that DNA binding was sufficient for TrpY repression of trpY transcription but that TrpY must bind DNA and tryptophan to assemble a complex that represses trpEGCFBAD.

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.

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.

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.

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.

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.