The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB.

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship.

Feast/famine regulation by transcription factor FL11 for the survival of the hyperthermophilic archaeon Pyrococcus OT3.

Transcriptional repressor FL11 from the hyperthermophilic archaeon, Pyrococcus OT3, was crystallized in its dimer form in complex with a DNA duplex, TGAAAWWWTTTCA. Chemical contacting of FL11 to the terminal 5 bps, and DNA bending by propeller twisting at WWW confirmed specificity of the interaction. Dimer-binding sites were identified in promoters of approximately 200 transcription units coding, for example, H+-ATPase and NAD(P)H dehydrogenase. In the presence of lysine, four FL11 dimers were shown to assemble into an octamer, thereby covering the fl11 promoter. In the "feast" mode, when P. OT3 grows on amino acids, the FL11 octamer will terminate transcription of fl11, as was shown in vitro, thereby derepressing transcription of many metabolic genes. In the "famine" mode in the absence of lysine, approximately 6000 FL11 dimers present per cell will arrest growth. This regulation resembles global regulation by Escherichia coli leucine-responsive regulatory protein, and hints at a prototype of transcription regulations now highly diverged.

A structural code for discriminating between transcription signals revealed by the feast/famine regulatory protein DM1 in complex with ligands.

Feast/famine regulatory proteins (FFRPs) comprise the largest group of archaeal transcription factors. Crystal structures of an FFRP, DM1 from Pyrococcus, were determined in complex with isoleucine, which increases the association state of DM1 to form octamers, and with selenomethionine, which decreases it to maintain dimers under some conditions. Asp39 and Thr/Ser at 69-71 were identified as being important for interaction with the ligand main chain. By analyzing residues surrounding the ligand side chain, partner ligands were identified for various FFRPs from Pyrococcus, e.g., lysine facilitates homo-octamerization of FL11, and arginine facilitates hetero-octamerization of FL11 and DM1. Transcription of the fl11 gene and lysine synthesis are regulated by shifting the equilibrium between association states of FL11 and by shifting the equilibrium toward association with DM1, in response to amino acid availability. With FFRPs also appearing in eubacteria, the origin of such regulation can be traced back to the common ancestor of all extant organisms, serving as a prototype of transcription regulations, now highly diverged.