Cox K H, Rai R, Distler M, Daugherty J R, Coffman J A, Cooper T G
Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163, USA.
J Biol Chem. 2000 Jun 9;275(23):17611-8. doi: 10.1074/jbc.M001648200.
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.
酿酒酵母通过抑制转运和分解代谢不良氮源所需基因的GATA因子依赖性转录，优先选择性地使用优质氮源（谷氨酰胺）而非劣质氮源（脯氨酸），这一生理过程称为氮代谢物阻遏（NCR）。我们发现一些对NCR敏感的基因（CAN1、DAL5、DUR1,2和DUR3）产生两种大小略有不同的转录本。较短转录本的合成对NCR敏感，而较长转录本的合成则不敏感。无论提供何种氮源，较长转录本在gln3Δ突变体中也占主导地位。我们证明，较长的mRNA种类是通过使用由Gln3p结合位点（GATAAs）产生的替代转录起始位点产生的，这些位点能够充当替代TATA元件。GATAAs作为替代TATA的能力，即当较短的、对NCR敏感的转录本的合成受到抑制时，与增强型绿色荧光蛋白（EGFP）-Gln3p在细胞质中的隔离相关，其方式与EGFP-Ure2p的情况无法区分。然而，当较短的、对NCR敏感的DAL5转录本占主导时，EGFP-Gln3p位于细胞核中。这些数据表明，NCR的潜在机制涉及Ure2p与Gln3p在细胞质中的结合，这种相互作用阻止Gln3p到达其在NCR敏感基因上游的结合位点。
