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.

A SELEX study of the DNA-binding specificity of archaeal FFRPs: 2. FL4 (pot1613368).

The DNA-binding specificity of a transcription factor, the FFRP FL4 (pot1613368) from Pyrococcus sp. OT3, was studied. Using SELEX (systematic evolution of ligands by exponential environment) experiments, from a set of fragments, ∼150 bps, of the genomic DNA of P. OT3, seven were selected as containing binding sites. Thirteen bases identified as shared by the seven selected fragments with the least mismatches, 2.71 on average, was ATGAA AAAGTCAT. This sequence was closely related with another sequence, ATGAA[AAA/TTT]TTCAT, in the 5-3-5 arrangement, i.e. NANBNCNDNE [AAA/TTT]NENDNCNBNA , where, e.g. NA was the base complementary to NA. The average number of mismatches found between this sequence and the seven fragments was 3.14. A sequence, TTGAA ATT TACAA, resembling the sequence ATGAA[AAA/TTT]TTCAT and also another 5-3-5 sequence, TTGAA[AAA/TTT]TTCAA, was found upstream of the fl4 gene, which is potentially recognized by FL4 for auto-regulation. Thus it is likely that an ideal binding-site of FL4 is ATGAA[AAA/TTT]TTCAT or TTGAA[AAA/TTT]TTCAA. In this abstract, the sequences were highlighted in Italic at 3, and with bold characters at 5 and 5. When two sequences compared were the same at some positions, there they were underlined.