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

Archaea, constituting the third primary domain of life, harbor a basal tran scription apparatus of the eukaryotic type, where:ls curiously, a large fra ction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicte d 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 arc haeal homologue of the Escherichia coli leucine-responsive regulatory prote in Lrp, a global transcriptional regulator and genome organizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the statio nary-growth phase and produced in smaller amounts in complex medium, this d own regulation being leucine independent, We report on Sa-Lrp protein purif ication from S. acidocaldarius and from recombinant E. coli, both identifie d by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was s hown to be homotetrameric and to bind to its own control region; this bindi ng proved to be leucine independent and was stimulated at high temperatures . Interference binding experiments suggested an important role for minor gr oove recognition in the Sa-Lrp-DNA complex formation, and mutant analysis i ndicated the importance for DNA binding of the potential helix-tnm-helix mo tif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purifi ed 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.