Processivity of Phage SP6 RNA Polymerase transcription

In the in vitro transcription reactions limiting concentrations of a ribonucleotide cause the phage SP6 RNA polymerase to pause long enough specifically only at the positions of the limited nucleotide and dissociate from the elongation complex. As a result a series of RNA oligomers comprising a sequencing ladder was obtained in high-resolution polyacrylamide-urea gel electrophoresis. Also, RNA oligomers up to 6-mer were made not only nucleotide-specifically, but also non-specifically. According to these results, the phage SP6 RNA polymerase appears to escape from the abortive initiation cycling after synthesizing 6 base long RNA, then get into the stable elongation mode as 7 base long RNA is made

Mutational Analysis of Transcription initiation Site Selection by Phage SP6 RNA Polymerase

The transcription initiation sites of the mutants containing various sequences around the initiation site of phage SP6 promoter were determined. Precise sizing of the abortive elongation product RNAs from the nucleotide-specific pausings of the phage SP6 RNA polymerase under nucleotide-limiting conditions determined the initiation site of each mutant. When the wild-type +1 G is changed to C or A without change in the upstream sequence including TATA from -4 to -1, it still starts only at the + 1 site. Therefore, it seems that the phage SP6 RNA polymerase selects the initiation site precisely at a certain distance from a direct contact point in the upstream promoter sequence, regardless of the species of initiating nucleotide. But, the mutant containing TATCC from -4 to + 1 starts transcription at both positions -1 C and + 1 C. This shift of the initiation site seems to be caused by the sequence-dependent perturbations of DNA helical structure, D form to B form

Study on the Regeneration of PCB Etchants in a Continuous Electrolytic Cell With On-Off Control System

A continuous electrolytic cell was used to regenerate the etching solution and recover a copper in a waste solution from the etching process of printed circuit boards(PCB). Diffusion coefficients of copper ions in 4M HCI solution were measured with a rotating disc electrode and the values are 2.21× 10⁻⁵㎠/sec for Cu(I) and 1.147× 10⁻⁵ ㎠/sec for Cu(II).Cu(II) forms complexes with chloride ions and then hydrated, and it results in the increase of radius and diffuses slowly. Dendritic copper deposition was observed above 300 mA/ ㎠ current density and the dendritic form is favorable to recover because it can be readily cleaned and detached from Ti cathode. Cu(I) oxidation took place in the absense of Cl2 evolution by controlling the inlet flow rate with in-situ measurement of potential(vs. Ag/AgCl/sat.KCI) of anolyte. On-off control sysstem for a continuous electrolytic cell was self-designed and optimally applied to regenerate the etching solution and copper by controlling the variables such as specific gravity, flow rate, and Cu(II)/Cu(I) ratio. PCB에칭 폐액으로부터 에칭액을 재생하고 구리를 회수하기 위하여 연속식 전해조를 제작 및 운영하였다. RDE(rotating disc electrode)를 통한 4M HCI 용액내에서의 구리이온의 확산을 조사하였으며, Cu(Ⅱ)의 확산계수는 1.147× 10⁻⁵ ㎠/sec 이며, Cu(I)에 대하여는 2.21× 10⁻⁵㎠/sec으로 Cu(II)의 확산이 Cu(I)보다 느리게 일어난다. 이는 Cu(II)가 염소이온을 함유한 용액내에서 착물을 이룬 상태에서 수화되므로 이온의 크기가 증가하기 때문으로 추측된다. Ti전극 위에서 300 mA/ ㎠의 전류밀도 이상에서 dendrite형태로 구리의 석출이 일어나며, Ti전극과의 분리가 쉽고 세척이 용이한 점을 고려할 때 금속상태로 구리를 회수시에 porous 형태보다 유리하였다. 양극실에서의 Cu(I)의 산화시 최소유속 이상으로 에칭폐액을 공급하면서 전해도중 연속적으로 양극전해질의 전위를 측정하여 유속을 on-off방식으로 조절함으로써 염소 가스 발생을 억제할 수 있었다. 그리고 연속식 전해조의 on-off 방식의 제어장치를 직접 제작하여 전해도중에 비중, 유량 및 Cu(II)/Cu(I)비 등을 제어함으로써 PCB에칭 폐액을 연속적으로 안정하게 재생하고 용액내의 구리를 회수할 수 있었다.본 연구는 선도기술개발사업에 의해 수행되었으며, 지원에 감사드립니다

파아지 SP6 RNA 중합효소의 전사개시위치

학위논문(석사) - 한국과학기술원 : 생물공학과, 1988.2, [ vi, 51 p. ]The transcription initiation sites of the phage SP6 initiation sequence mutants containing deletions around the wild-type initiation site were determined. It could be possible to obtain sequencing ladders in high-resolution polyacrylamide-urea gel electrophoresis, using the phage SP6 RNA polymerase under the in vitro transcription conditions where the limiting concentration of a ribonucleotide causes the polymerase to pause long enough and terminate at the positions of the nucleotide. Precise sizing of the transcripts produced by this abortive elongation determined the initiation site of each mutant. When the wild-type position +1 G is changed to C, it still starts at the C. Also the wild-type position +1 G is changed to A, it still starts at the A. But, the mutant containing CCC sequence from -1 to +2 starts transcription at the C at both the wild-type position -1 and +1 with equal frequency.한국과학기술원 : 생물공학과

인체 미토콘드리아의 RNA 중합효소와 전사 종결 인자

학위논문(박사) - 한국과학기술원 : 생물과학과, 2001.2, [ v, 109 p. ]PART 1 The recombinant protein of the catalytic subunit of human mitochondrial RNA polymerase (h-mtRP) was overexpressed in Escherichia coli., and was purified to near homogeneity. The recombinant h-mtRP shows the same characteristics of the partially purified native enzymes from human mitochondria. The recombinant h-mtRP with recombinant h-mtTFA lacks a promoter-dependent transcription activity, pointing to another component(s) being required for promoter-dependent transcription. The promoter-specific binding of recombinant h-mtRP was observed using the electrophoretic mobility shift assay. The binding affinity of h-mtRP for LSP promoter was much greater than that for the HSP promoter, and was independent to the presence of the mtTFA. The binding of mtTFA to the promoters was also not dependent on the presence of the h-mtRP. The tertiary complexes containing both the mtTFA and h-mtRP bound to promoter sequences were observed. The h-mtRP is shown to possess the novel ability to direct robust but promiscuous transcription with mtTFA on duplex DNA templates with 3’-adenosine protruding ends. Analysis of reaction products indicates that h-mtRP, only with mtTFA, is capable of extending the 3’-termini of duplex DNA having 3’-adenosine-protruding DNA ends by ribonucleotides addition. Based on these findings, it is proposed that h-mtTFA activates the h-mtRP by helping to add the following RNA chains to the 3’-adenosine-terminus of the nascent RNA chain. The missing component for the promoter-directed transcription would be a protein to facilitate the h-mtRP to melt the DNA and to start the initial RNA synthesis. PART2 Transcription termination of human mitochondrial genome requires the action of a site-specific DNA-binding protein that binds to a single location of the entire human mitochondrial genome. A specific DNA-binding protein (mTERF) for the termination sequence was recently been identified and cloned. To study the functional characteristi...한국과학기술원 : 생물과학과

Regeneration of PCB Etchants and Copper Recovery in a Batch - type Electrolytic Cell

인쇄회로기판의 식각폐수를 전기화학적 방법을 이용하여 양극에서 이를 재생하고, 음극에서 구리를 석출하기 위한 실험을 행하였다. 양극에서의 Cu(I)의 산화에 따른 Cu(I)/Cu(Ⅱ) 변화는 Pt와 Ag/AgCl/4M KCl 전극사이의 전위차를 이용하여 측정하였으며, 반응의 진행에 따른 양극에서의 염소기체 발생은 용액내에 Cu(I)의 농도를 일정치 이상으로 유지시키고, 비다공성 흑연전극을 이용하여 억제할 수 있었다. 그리고, 음극에서의 구리석출은 전류밀도 360㎃/㎠, 구리이온농도 12g/ℓ 일때 가장 효율적이며 석출된 구리는 dendrite구조임을 알 수 있었다. 또한 석출효율과 회수방법을 고려할 때 음극으로서 Ti전극을 사용할 경우 가장 우수한 효율을 얻을 수 있었다. 전해온도가 증가함에 따라서 전류효율은 낮아졌으며, 전력효율은 50℃에서 최대값을 나타내었다. Anodic regeneration of PCB enchant and cathodic deposition of copper using electrochemical method has been studied. Cu(I)/Cu(Ⅱ) concentration ratio as a function of Cu(I) oxidation at the anode was measured from the potential difference between platinum and Ag/AgCl/4M KCl electrodes. Chlorine gas evolution was minimized by maintaining Cu(I) concentration above a specific concentration and using non-porous graphite electrode. Dendritic copper deposition was observed at the cathode and the optimum conditions for Cu deposition was identified as the current density of 360㎃/㎠, and copper concentration of 12 g/ℓ. Titanium was the most effective cathode material which showed a higher current efficiency and copper recovery. The current efficiency decreased with increasing temperature, but the highest power efficiency was achieved at 50℃.본 연구는 환경부 및 통상산업부지원 선도기술개발사업(G7)의 일환으로 수행되었으며, 연구비를 지원해 주신 삼성전기(株)에 감사드립니다