Cell cycle-dependent transcription of CLN2 is conferred by multiple distinct cis-acting regulatory elements.
The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation. CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1. We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation. Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment. This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs). Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription. Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity. We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter. Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28. This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs. Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function. The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation.
Stuart
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Scripps Research Institute, La Jolla, California 92037.
Wittenberg
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D001483:Base Sequence; D002453:Cell Cycle; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000096:biosynthesis; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic
C082721:CLN2 protein, S cerevisiae; D016213:Cyclins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012333:RNA, Messenger; C062214:SWI4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors