ABSTRACT
The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4(1)22, with unit-cell parameters a = b = 104.5, c = 245.1 A. Consideration of the values of V(M) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules.
Subject(s)
Archaeal Proteins/chemistry , DNA-Binding Proteins/chemistry , Pyrococcus furiosus/chemistry , Transcription Factors/chemistry , Crystallization , Crystallography, X-Ray , Leucine-Responsive Regulatory Protein , Protein Conformation , Protein Structure, QuaternaryABSTRACT
Transcription from many archaeal promoters can be reconstituted in vitro using recombinant TATA-box binding protein (TBP) and transcription factor B (TFB)--homologues of eukaryal TBP and TFIIB--together with purified RNA polymerase (RNAP). However, all archaeal genomes sequenced to date reveal the presence of TFE, a homologue of the alpha-subunit of the eukaryal general transcription factor, TFIIE. We show that, while TFE is not absolutely required for transcription in the reconstituted in vitro system, it nonetheless plays a stimulatory role on some promoters and under certain conditions. Mutagenesis of the TATA box or reduction of TBP concentration in transcription reactions sensitizes a promoter to TFE addition. Conversely, saturating reactions with TBP de-sensitizes promoters to TFE. These results suggest that TFE facilitates or stabilizes interactions between TBP and the TATA box.
Subject(s)
Archaeal Proteins/metabolism , DNA-Binding Proteins/metabolism , Sulfolobus/genetics , TATA Box/genetics , Transcription Factors, TFII , Transcription Factors/metabolism , Transcription, Genetic , Amino Acid Sequence , Archaeal Proteins/chemistry , Base Sequence , DNA Footprinting , DNA-Directed RNA Polymerases/metabolism , Deoxyribonuclease I/metabolism , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Subunits , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , TATA-Box Binding Protein , Transcription Factors/chemistryABSTRACT
The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine-responsive regulatory protein is the archetype. Its crystal structure has been determined at 2.9 A resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile. The structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed alpha/beta character reminiscent of a number of RNA- and DNA-binding domains. Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel beta-sheets of the C-terminal domain, and further interactions lead to octamer formation. The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression. A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.
Subject(s)
Archaeal Proteins/chemistry , DNA-Binding Proteins/chemistry , Pyrococcus furiosus/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Binding Sites , Calorimetry, Differential Scanning , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Escherichia coli Proteins , Gene Expression Regulation, Archaeal , Leucine/genetics , Leucine/metabolism , Leucine-Responsive Regulatory Protein , Models, Molecular , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Pyrococcus furiosus/genetics , Sequence Alignment , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.
Subject(s)
DNA, Archaeal/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Archaeal , Promoter Regions, Genetic , Pyrococcus furiosus/genetics , Pyrococcus furiosus/metabolism , Transcription Factors/genetics , Transcription, Genetic , Amino Acid Sequence , Archaeal Proteins , Base Sequence , Binding Sites , DNA, Archaeal/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli , Leucine-Responsive Regulatory Protein , Molecular Sequence Data , Nucleic Acid Conformation , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
A single point mutation in the 23S rRNA gene of Helicobacter pylori is known to confer resistance to clarithromycin. Most prevalent among clarithromycin-resistant clinical H. pylori isolates are the mutations from A-2142 to G and A-2143 to G in the 23S rRNA gene. The bias in the 23S rRNA gene mutations conferring clarithromycin resistance may result from the higher MIC, stability of resistance, and growth rate found for the strains with the above-mentioned mutations.
