Abstract
Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.
Highlights
Arginine transport into Saccharomyces cerevisiae is mediated by several permeases; one is the low Km (10 mm) basic amino acid permease responsible for arginine uptake at low external concentrations
We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements
The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, nitrogen catabolite repression (NCR)-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p
Summary
Arginine transport into Saccharomyces cerevisiae is mediated by several permeases; one is the low Km (10 mm) basic amino acid permease responsible for arginine uptake at low external concentrations. Gap1p is produced only in medium containing a poor nitrogen source [2, 3] These early studies gave rise to the accepted notion that CAN1 expression was insensitive to nitrogen catabolite repression (NCR).. NCR-sensitive transcription of genes required for uptake and catabolism of poor nitrogen sources is mediated by UASNTR elements [4, 5]. UASNTR, referred to as GATA elements since they contain the core sequence GATAA, are the binding sites for the NCR-sensitive transcriptional activators Gln3p and Gat1p/Nil1p [4, 5]. The critical observation was that heterologous overproduction of Can1p (driven by the ADH1 promoter) resulted in arginine-induced CAR1 expression becoming insensitive to NCR [14]. Utilization of an upstream start site accounts for the increased size of the NCR-insensitive tran-
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