DksA Guards Elongating RNA Polymerase against Ribosome-Stalling-Induced Arrest

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Mutagenic Specificity of a Novel T4 DNA Polymerase Mutant

The in vivo mutational specificity of a novel T4 DNA polymerase mutator mutant, tsM19, was determined. Two genetic tester systems were used to characterize the mutant. Results of our studies indicate that tsM19 promotes transition and transversion mutagenesis and, possibly, frameshift mutagenesis. Central G:C base pairs in runs of three or more consecutive G:C base pairs may be target sites for tsM19-induced transitions.     

Altered Deoxyribonucleic Acid Polymerase Activity in a Methyl Methanesulfonate-Sensitive Mutant of Bacillus subtilis

Of six deoxyribonucleic acid repair mutants of Bacillus subtilis assayed for deoxyribonucleic acid polymerase, only the methyl methanesulfonate-sensitive and ultraviolet light-sensitive mutant JB1-49(59) has impaired polymerase activity. Extracts prepared by sonic treatment or gentle lysis had about 10% of the wild-type activity with poly d(A-T), an alternating copolymer of deoxyadenylate and deoxythymidylate, used as template. The sensitivity to methyl methanesulfonate and ultraviolet light and the low level of polymerase activity transformed and reverted together, indicating that the two characteristics are a pleiotropic manifestation of a single mutation. Mixed extract and kinetic experiments mitigated against an altered nuclease activity as the enzymatic consequence of the mutation. Also, the mutant and wild type activities were stimulated equally by Escherichia coli exonuclease III. The residual activity in the mutant showed several differences from the wild-type activity: it purified differently, was more sensitive to sulfhydryl reagents, and displayed a different template specificity. We tentatively conclude that either the mutation in JB1-49(59) has introduced a qualitative as well as a quantitative change in the polymerase or the wild type contains two distinct polymerases, one of which is missing in the mutant.     

Poly A Synthesizing Activity in a Constitutive Subunit of RNA Polymerase

The α-like subunit in RNA polymerase was demonstrated to polymerize poly A from ATP. The enzymatic character and possible role of the poly A polymerization activity in the RPase are discussed.     

棉铃虫5型质型多角体病毒RNA聚合酶的确定与功能机制研究

棉铃虫5型质型多角体病毒属于呼肠孤病毒科质型多角体病毒属,以重要农业害虫棉铃虫为其天然宿主,对棉铃虫的生物控制具有重要意义.本文对棉铃虫5型质型多角体病毒第3片段编码的蛋白的功能进行了初步研究.首先通过同源性对比,推测其所编码的蛋白可能行使RNA依赖的RNA聚合酶(RdRP)的功能.通过体外活性研究确定了该蛋白的RdRP活性,并确定了其保守活性位点GDD.随后以病毒基因组RNA和3′-OH封闭的病毒基因组RNA为模板,利用Northern blot方法研究该蛋白起始病毒基因组RNA合成的分子机制.结果表明,该病毒的RdRP主要通过引物非依赖的方式起始病毒基因组RNA的合成,并且该RdRP蛋白并不具有末端转移酶活性.最后,对RdRP行使功能的生化条件进行探索,发现RdRP功能的发挥需要二价金属离子Mg2+的存在.     

Effect of Ischemia on the Activity of DNA-Dependent RNA Polymerase and DNA Polymerase

Abstract: Ischemia, anoxia, and hypoxia of the brain have been shown to inhibit protein synthesis in the central nervous system. To obtain data on the changes in DNA-dependent RNA and DNA polymerases as they pertain specifically to neurons and glia, nuclear enriched neuronal and glial fractions were prepared, by sucrose-gradient centrifugation, from spinal cords of adult dogs that had been subjected to prolonged ischemia. The isolated fractions were assayed for enzyme activity by a radiochemical technique. RNA polymerase was affected more than DNA polymerase, activity being reduced considerably in both neurons and glia. Possible causes of the difference in sensitivity to ischemia are discussed.     

Deoxyribonucleic Acid Polymerase Activity in a Deoxyribonucleic Acid Polymerase I-Deficient Mutant of Bacillus subtilis Infected with Temperate Bacteriophage SPO2

Increased deoxyribonucleic acid (DNA) polymerase activity is found in soluble extracts from a polymerase I-negative mutant of Bacillus subtilis after infection with temperate phage SPO2, or after induction of SPO2 prophage in lysogenic derivatives of this mutant. No increased enzyme activity is found after SPO2 infection in the presence of chloramphenicol. Infection of the polymerase-negative mutant with the DNA-negative sus mutant SPO2 L244 gives no increased enzyme activity, whereas infection with DNA-negative sus mutant SPO2 J385 gives enzyme activities comparable to those found in wild-type infected cells. These findings suggest that SPO2 determines a DNA polymerase activity essential for synthesis of phage DNA.     

Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

We have purified and characterized a DNA-dependent RNA polymerase from the blue-green alga Fremyella diplosiphon. This enzyme, purified by gel filtration, DEAE-cellulose chromatography, and glycerol gradient centrifugation, is comprised of five polypeptide subunits. Their masses are 161,000, 134,000, 91,000, 72,000, and 41,000 daltons. Preparative electrophoresis of the purified enzyme on nondenaturing gels separates the 41,000-dalton polypeptide from the rest of the enzyme. The enzyme is extremely labile in the presence of a variety of salts of strong acids and bases; the purification procedure was devised to avoid exposure to such compounds.