|Budget Amount *help
¥13,200,000 (Direct Cost : ¥13,200,000)
Fiscal Year 1995 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1994 : ¥10,200,000 (Direct Cost : ¥10,200,000)
This project aimed to establish the imaging methods of transcription process of DNA,especially three dimensionalassembly of DNA,RNA polymerase and regulatory proteins complex. For this purpose, we developed first freeze-drying apparatus by molecular distilllation, that is available for atomic force imaging. Howefver, atomic force microscope was not able to described molecular architecture of DNA-protein complex. rather classical low angle shadowing detected obviously the complex structure. To improve the resolution of shadowing method, we developed new freeze etching machine working at extremely high vacuum (10^<-6> P). Platinum particles evaporated in this apparatus at-100ﾟC was so small that basic structure of DNA as well as molecular organization of DNA binding proteins such as polymerase was depicted by low angle rotary shadowing.therefore, we used this technique exclusively for imaging of DNA transcription processes. High resolution rotary shadowing revealed that E.coli RNA polymerase consisted of 5 subunits which is well consistentwith previous biochemical evidence. Depending upon molecular weight of subunits, structures corresponding to alpha, beta, gamma subunits were identified respectively. alpha, alpha andbeta, beta'subunit formed two channels. One of them, larger one, was located between 2 alphaandbeta, beta'. The another one was found between beta and beta'like a slit. Assembled DNA was always observed in the groove between 2alpha and beta, beta'. These are suggested that newlysynthesized mRNA may come up along the slit formed by betaandbeta'.