Curcio M J, Garfinkel D J
Laboratory of Eukaryotic Gene Expression, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201.
Mol Cell Biol. 1992 Jun;12(6):2813-25. doi: 10.1128/mcb.12.6.2813-2825.1992.
High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.
在2微米质粒(pGTy1)上与可诱导的GAL1启动子融合的具有转座活性的Ty1元件的高水平表达克服了酿酒酵母中的转座休眠。为了研究控制Ty1逆转录转座速率的机制,我们对诱导和未诱导pGTy1表达的细胞中的转座和Ty1基因产物进行了定量。Ty1转座的增加比pGTy1诱导所影响的Ty1 RNA的增加大45至125倍。在转座诱导的细胞中,Ty1 RNA的翻译效率没有改变,因为p190TYA1-TYB1蛋白合成与稳态Ty1 RNA水平成比例增加。因此,pGTy1元件的表达在翻译后水平提高了Ty1转座的效率。pGTy1的半乳糖诱导增强了TYA1蛋白的加工,并使得能够检测到加工后的TYB1蛋白,这些蛋白在未诱导的细胞中通常以非常低的水平存在。当检查基因组Ty1元件对HIS3标记的pGTy1元件中特定突变的互补能力时,蛋白酶结构域中的突变或整合酶结构域中的某些突变未能得到互补,但逆转录酶结构域中的突变被基因组Ty1元件部分互补。因此,酵母细胞中Ty1元件的活性可能受到Ty1蛋白酶以及可能的整合酶可用性的限制。这些结果表明,Ty1转座在蛋白质加工水平上受到调控,并且这种调控通过pGTy1元件的表达得以克服。