Abstract：
Maf1 is a negative regulator of RNA polymerase III (Pol III) in yeast. Maf1-depleted cells manifest elevated tRNA transcription and inability to grow on non-fermentable carbon source, such as glycerol. Using genomic microarray approach, we examined the effect of Maf1 deletion on expression of Pol II-transcribed genes in yeast grown in medium containing glycerol. We found that transcription of FBP1 and PCK1, two major genes controlling gluconeogenesis, was decreased in maf1Δ cells. FBP1 is located on chromosome XII in close proximity to a tRNA-Lys gene. Accordingly we hypothesized that decreased FBP1 mRNA level could be due to the effect of Maf1 on tgm silencing (tRNA gene mediated silencing). Two approaches were used to verify this hypothesis. First, we inactivated tRNA-Lys gene on chromosome XII by inserting a deletion cassette in a control wild type strain and in maf1Δ mutant. Second, we introduced a point mutation in the promoter of the tRNA-Lys gene cloned with the adjacent FBP1 in a plasmid and expressed in fbp1Δ or fbp1Δ maf1Δ cells. The levels of FBP1 mRNA were determined by RT-qPCR in each strain. Although the inactivation of the chromosomal tRNA-Lys gene increased expression of the neighboring FBP1, the mutation preventing transcription of the plasmid-born tRNA-Lys gene had no significant effect on FBP1 transcription. Taken together, those results do not support the concept of tgm silencing of FBP1. Other possible mechanisms are discussed.
摘要：
Maf1是酵母中RNA聚合酶III(PolIII)的负调节因子。Maf1耗尽的细胞显示tRNA转录升高，无法在不可发酵的碳源上生长，如甘油。使用基因组微阵列方法，我们检查了Maf1缺失对在含甘油的培养基中生长的酵母中PolII转录基因表达的影响。我们发现控制糖异生的两个主要基因FBP1和PCK1的转录，在maf1Δ细胞中减少。FBP1位于染色体XII上,紧邻tRNA-Lys基因。因此,我们假设降低的FBP1mRNA水平可能是由于Maf1对tgm沉默(tRNA基因介导的沉默)的影响。使用两种方法来验证这一假设。首先,我们通过在对照野生型菌株和maf1Δ突变体中插入缺失盒来灭活XII染色体上的tRNA-Lys基因。第二,我们在与质粒中相邻的FBP1克隆的tRNA-Lys基因的启动子中引入了点突变，并在fbp1Δ或fbp1Δmaf1Δ细胞中表达。通过RT-qPCR测定每个菌株中FBP1mRNA的水平。尽管染色体tRNA-Lys基因的失活增加了邻近FBP1的表达，但阻止质粒出生的tRNA-Lys基因转录的突变对FBP1转录没有显着影响。一起来看,这些结果不支持FBP1的tgm沉默的概念。讨论了其他可能的机制。
