Regulation of the cycling of timeless (tim) RNA

Circadian rhythms in Drosophila depend upon expression of the timeless (tim ) and period (per) genes, which encode interacting components of the endoge nous clock. These two clock genes show a robust circadian oscillation in tr anscription rate as well as RNA, and protein levels. Transcriptional activa tion of both genes requires the basic helix-loop-helix (bHLH) PAS transcrip tion factors dCLOCK (dCLK) and CYCLE: (CYC), which bind E-box elements. We investigated the role of E-box elements in regulating behavioral rhythmicit y and tim gene expression. We show that mutation of the upstream E-box in t he tim gene prevents the rescue by tim cDNA sequences of the arrhythmic tim (01) phenotype, RNA encoded by this mutated tim transgene fails to cycle an d is expressed at low levels, While a tint transgene carrying a wild-type E -box restores behavioral rhythms, tim RNA levels are intermediate to those of the mutant E-box transgenic lines and wild type, and do not display high amplitude cycling. On the other hand, high-amplitude RNA cycling was consi stently obtained with a tim transgene that contains genomic, rather than cD NA, sequences. To identify additional sequences that may be required for ti m cycling, we investigated the role of an E-box in the first intron of the tim gene through cell culture experiments. In these experiments, the presen ce of this intron did not have any effect on the activation of tim transcri ption by dCLK/CYC. As the upstream E-box was implicated in activation by dC LK/CYC in cell culture, we assayed sequences containing this E-box for asso ciation with proteins in fly head extracts. These studies provide the first biochemical evidence for an in vivo complex containing dCLK and CYC that b inds the tim upstream sequence and is detected at all times of day. Togethe r, these data highlight molecular mechanisms that are critical for behavior al rhythms. (C) 2001 John Wiley & Sons. Inc.