Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Identification of a DNA supercoiling activity in Saccharomyces cerevisiae.

A relaxed plasmid DNA is shown to become positively supercoiled in cell extracts from top1 strains of Saccharomyces cerevisiae. This positive supercoiling activity is dependent on the presence of bacterial DNA topoisomerase I and ATP (or dATP), and the positive supercoils generated in this reaction are not constrained by protein(s). Non-hydrolyzable ATP analogs cannot substitute for ATP in this supercoiling reaction, and the supercoiling activity is not due to RNA synthesis. The presence of an ARS sequence in the DNA does not alter the activity. Furthermore, this activity is equally active against UV irradiated or intact DNA. Extracts prepared from rad50 and rad52 mutant cells exhibited the same activity. Partial purification of this activity suggests that a protein factor with a native molecular weight of approximately 150 kDa is primarily responsible for the activity. The possibility that this supercoiling activity may be due to tracking of a protein along the intact duplex DNA is discussed.     

Rifampin and rpoB mutations can alter DNA supercoiling in Escherichia coli.

Two cases are described which indicate that RNA polymerase could alter DNA supercoiling. One occurred in a topA mutant in which abnormally high levels of plasmid supercoiling were lowered by rifampin, an inhibitor of the beta subunit of RNA polymerase. The second case involves suppression of a temperature-sensitive gyrB mutation by a rifampin-resistant allele of rpoB, the gene encoding the beta subunit of RNA polymerase. Measurements of chromosomal DNA supercoiling show that the rpoB mutation reduced DNA relaxation.     

Interrelated effects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter

The formation of complexes containing high levels of DNA melting at the ribosomal RNA rrnB P1 promoter in vitro is shown to be facilitated by DNA supercoiling or low salt. The effector nucleotide ppGpp is ineffective under these conditions. The loss of supercoils or addition of salt increases the effectiveness of ppGpp in inhibiting formation of these complexes. In vivo plasmid DNA supercoiling is shown to decrease during starvation protocols that also increase levels of ppGpp. The results suggest that ppGpp regulation may be affected by the state of DNA supercoiling in vivo.     

R环的形成及对基因组稳定性的影响

R-环是由一个RNA:DNA杂交体和一条单链状态的DNA分子共同组成的三链核酸结构。其中, RNA:DNA杂交体的形成起因于基因转录所合成的RNA分子不能与模板分开, 或RNA分子重新与一段双链DNA分子中的一条链杂交。在基因转录过程中, 当转录泡遇到富含G碱基的非模板链区或位于某些与人类疾病有关的三核苷酸卫星DNA时, 转录泡后方累积的负超螺旋可促进R环形成。同时, 新生RNA分子未被及时加工、成熟或未被快速转运到细胞质等因素也会催生R环。研究表明, 细胞拥有多种管理R环的方法, 可以有效地管理R环的形成和处理已经形成的R环, 以尽量避免R环对DNA复制、基因突变和同源重组产生不利影响。文章重点分析了R-环的形成机制及R环对DNA复制、基因突变和同源重组的影响, 并针对R-环诱导的DNA复制在某些三核苷酸重复扩增有关的神经肌肉退行性疾病发生过程中的作用进行了分析和讨论。     

Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli.

Incubating cultures of Escherichia coli with propionic acid (5 mmol/l) or formic acid (10 mmol/l) at pH 5.0 produced bacteriostasis lasting 30 and 120 min respectively. During this time rates of RNA, DNA, protein, lipid and cell wall synthesis were reduced. Growth resumed after continued incubation in the presence of acid, but cells from acid-treated cultures were larger than controls. DNA synthesis was particularly sensitive to the presence of the propionic or formic acid.     

Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli

Incubating cultures of Escherichia coli with propionic acid (5 mmol/l) or formic acid (10 mmol/l) at pH 5.0 produced bacteriostasis lasting 30 and 120 min respectively. During this time rates of RNA, DNA, protein, lipid and cell wall synthesis were reduced. Growth resumed after continued incubation in the presence of acid, but cells from acid-treated cultures were larger than controls. DNA synthesis was particularly sensitive to the presence of the propionic or formic acid.