Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound ("engaged") and unbound ("free") forms revealed that the steroid-mediated inhibition of the activity of the "engaged" form of the enzyme was not accompanied by significant changes in "free" pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.     

Anabolic steroid metabolism in skeletal muscle

Three hundred and sixty male albino rats weighing 180 to 200 g were used to determine the effect of anabolic steroid hormones on adaptive changes in the synthesis of ribosomal RNA both in sedentary animals and in animals involved in a training programme. One injection of Retabolil (0.1 mg/100 g body weight) increased the α-amanitin insensitive RNA polymerase activity of nuclei from skeletal muscles. Fourteen h after this hormone injection the enzyme activity was 45% higher than in control animals and it remained at this level for 4 days. Under these conditions a selective binding of 19-nortestosterone with cytoplasmic proteins of skeletal muscle was found. Physical training increased the RNA polymerase activity by 50% (P < 0.05). It was found that the testosterone binding capacity of a cytoplasmic extract from trained animals was 70% greater than that of the control animals (P < 0.05). Four injections of Retabolil during training resulted in an additional increase of RNA polymerase activity of 40% (P < 0.05) but reduced the testosterone binding capacity of the cytoplasmic proteins that occurred with training by 21%. These results demonstrate the effect of anabolic hormones in the regulations of RNA synthesis in skeletal muscle nuclei in the process of their adaptation to systematic physical training.     

Phosphorylation of Escherichia coli RNA polymerase by rabbit skeletal muscle protein kinase

Rabbit skeletal muscle protein kinase catalyzes the phosphorylation of DNA-dependent RNA polymerase of Escherichia coli in the presence of adenosine 3′,5′-monophosphate and ATP. The phosphorylation occurs on one (or more) serine residue(s) in the σ-factor under reaction conditions similar to those employed for RNA synthesis. The phosphorylation of RNA polymerase and its stimulation by protein kinase are inhibited by a specific heat-stable inhibitor from rabbit skeletal muscle. With conditions more favorable for the protein kinase reaction, phosphorylation of RNA polymerase also occurs on the β subunit of the core enzyme, but this reaction occurs at a much slower rate than the phosphorylation of the σ-factor.     

USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.     

真核基因启动子非依赖的功能转录延伸复合物的体外组装

真核生物RNA聚合酶Ⅱ的持续合成能力对基因转录过程中每一个阶段,包括启动子脱离、转录暂停、转录终止以及转录偶联DNA损伤修复过程的调节至关重要.在RNA聚合酶Ⅱ介导的转录延伸过程中,其和模板DNA及转录产物RNA紧密结合,形成一个非常稳定的延伸三维复合物(elongationcomplex,EC).此特征性“泡”状结构的形成是RNA聚合酶Ⅱ持续合成能力所必需的.在不依赖启动子及众多转录起始因子的条件下,利用人工合成的RNA与DNA寡核苷酸,在体外组装形成具有功能转录活性的延伸复合物.结果表明,长度为9个核苷酸的RNA与模板DNA形成的杂合分子对转录延伸复合物的形成是必需的,而非转录模板DNA链的加入导致最终活性转录“泡”状复合物的形成,并可转录形成与模板相关的转录产物,进一步通过在模板DNA的特定位置引入一个乙酰氧乙酰氨基芴修饰基团,可特异性地阻断转录延伸过程,从而显示该系统在研究真核基因转录及转录偶联DNA损伤修复机制中的潜在应用价值.