Purification and characterization of Sa-lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcriptional regulator Lrp
| Autor: | Thia-Lin Thia-Toong, Daniel Gigot, Daniel Charlier, Nicolas Glansdorff, Julius J. Enoru-Eta |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Sulfolobus acidocaldarius
Operator Regions Genetic Archaeal Proteins Recombinant Fusion Proteins Molecular Sequence Data DNA Footprinting Gene Expression Genetics and Molecular Biology RNA Archaeal Ligands Microbiology DNA-binding protein Leucine-responsive regulatory protein Escherichia coli Deoxyribonuclease I RNA Messenger Molecular Biology Peptide sequence Transcription factor Gene General transcription factor biology Base Sequence Binding protein Escherichia coli Proteins Helix-Loop-Helix Motifs Temperature DNA Leucine-Responsive Regulatory Protein DNA-Binding Proteins DNA Archaeal Biochemistry Amino Acid Substitution Genes biology.protein lipids (amino acids peptides and proteins) Transcription Factors |
| Zdroj: | Journal of bacteriology. 182(13) |
| ISSN: | 0021-9193 |
| Popis: | Archaea , constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius , encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent. We report on Sa-Lrp protein purification from S. acidocaldarius and from recombinant E. coli , both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp–DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of l -leucine, suggesting a potential physiological role of the amino acid as a cofactor. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|