Analysis of frequency (frq) clock gene homologs: evidence for a helix-turn-helix transcription factor

Abstract

The frq gene plays a key role in the organization of the Neurospora crassa circadian clock. Our previous analysis of a C-terminal fragment of the putative FRQ protein suggested it is a nuclear transcription factor but did not identify a known DNA-binding domain. If the hypothesis is correct that FRQ is a transcription factor, sequences consistent with this function should be conserved in distantly related species. To investigate, we have cloned frq homologs from other filamentous fungi including Chromocrea spinulosa, and Leptosphaeria australiensis. Alignment of the Leptosphaeria and Chromocrea proteins with the published (complete) sequences for Neurospora crassa and Sordaria fimicola shows that they are respectively about 47% and 43% identical to both Neurospora and Sordaria. The alignment identifies several short regions of high conservation punctuated by regions showing near total divergence. Sequences consistent with FRQ being a transcription factor are generally conserved. Most importantly, we show that a highly conserved segment of the protein has strongly predicted helix-turn-helix (HTH) structure as supported by three independent methods. Further, this segment shows the defining sequence characteristics of known HTH DNA-binding domains. Amino acids at positions altered in frq mutant alleles are conserved in all species examined. Transformation of the Neurosporafrq ⁹(conditionally arrhythmic) mutant with the Chromocrea homolog rescued the pigmentation and conidiation defects of the mutant but not the circadian defect; the Leptosphaeria homolog failed to rescue any defect. Together, these data provide the first testable hypotheses concerning several specific aspects of FRQ structure and function.