Gilbert H J, Stephenson J R, Tully M
J Bacteriol. 1983 Mar;153(3):1147-54. doi: 10.1128/jb.153.3.1147-1154.1983.
The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.
研究了红酵母中编码苯丙氨酸解氨酶（PAL）的功能性mRNA的调控。含聚腺苷酸[poly(A)]的RNA是兔网织红细胞裂解物中体外翻译的有效模板。不含poly(A)的RNA不能刺激体外蛋白质合成。几条实验证据表明，红酵母的mRNA在网织红细胞裂解物中指导PAL的合成：（i）当与含酵母poly(A)的RNA一起孵育的裂解物与PAL抗血清反应时，免疫沉淀物中的主要放射性产物与天然PAL（75,000）具有相同的分子量；（ii）这种主要放射性产物与天然PAL竞争与PAL抗血清的结合；（iii）体外翻译产物的部分蛋白水解肽图与天然PAL的非常相似。当红酵母在不同生理条件下生长时，通过定量向网织红细胞裂解物中添加RNA时体外合成的PAL来确定编码PAL的功能性mRNA的水平。在以苯丙氨酸为生长底物的酵母中，功能性PAL mRNA比在葡萄糖-苯丙氨酸基本培养基中生长的酵母高6倍。在以葡萄糖-氨基本培养基中生长的酵母中，无论有无苯丙氨酸，均未检测到功能性PAL mRNA。观察到的功能性PAL mRNA的这些变化与PAL催化活性和抗原活性水平相似。研究了功能性PAL mRNA合成和降解的动力学。在转移到PAL诱导生长条件后60分钟内观察到功能性PAL mRNA的最高水平。当细胞从苯丙氨酸转移到葡萄糖-氨基本培养基时，含poly(A)的RNA和功能性PAL mRNA迅速降解，半衰期分别为25分钟和10分钟。因此，有人认为，在不同生理条件下生长的红酵母细胞中PAL量的变化主要是由于编码该酶的功能性mRNA量的改变。
