Figure 4.

uORFs restrict translation of FRQ. (A) In frqΔUTR residues, 50-1477 of the 5′-UTR are deleted and an AscI site is introduced. Western blot and densitometric quantification showing temperature-dependent expression of FRQ-encoded frqΔUTR. (B) Construction frq1/6^mut: Start codons of uORF1 and uORF6 were altered by site-directed mutagenesis to GTG. Temperature-dependent expression of FRQ encoded by frq and frq1/6^mut is shown. (C) The free-running period of frq1/6^mut (squares) and wild-type (circles) control (frq) was determined on race tubes at 25°C and 28°C. (D) Construction of ΔI-2 frq1/6^AUG and ΔI-2 frq1/6^mut: cDNA derived from I-2 spliced frq RNA was used as template for PCR reactions. Fragments including the translation initiation site of uORF1 and uORF6 were amplified by PCR and inserted into the AscI site of frqΔUTR. Start codons of uORF1 and uORF6 were either left unchanged (ΔI-2 frq1/6^AUG) or changed to TTG and ACG (ΔI-2 frq1/6^mut). In the constructs shown, the C terminus of uORF6 (... HKLLERE) was changed to... HKGAPSSIAGF. (E) frq6^cons: The translation initiation site of uORF6 was altered toward consensus (CAACATGG), and expression of FRQ was measured at the indicated temperatures. The ratios of FRQ expressed by frq6^cons vs. FRQ expressed by a corresponding wild-type frq allele are shown. (F) Conidiation of the strain frq6^cons. Race tubes were inoculated in LL and incubated at 25°C in DD. After 24 h, they were transferred to the indicated temperatures.