| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2002: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 2001: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
The mechanism of transcription initiation has been assumed to be a sequence of three essential steps: formation of a complex between RNA polymerase and a promoter (closed complex), formation of another complex with partially melted DNA duplex to form phosphodiester bonds (open complex and chemical reaction), and escape of RNA polymerase from the promoter associated with the progress of RNA elongation (promoter clearance). There is a process called "abortive initiation", an iterative synthesis and release of oligo-RNA molecules, and is often excluded from the mainstream of the mechanism. Altough this process has been observed in vitro with all prokaryotic and eukaryotic RNA polymerases so far isolated, its role as well as its occurrence in vivo has been unknown.
We have been clarifying for several years that this sequential mechanism is not the case and the initiation follows branched pathways, one of which contains the moribund complex, being defined as a complex that produces only abor
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tive and no full-length transcripts. Followings are its characteristics. 1.The moribund complex, as well as the productive complex that synthesizes full-length product, are formed from the same homogeneous fraction of enzyme molecules, and dissociation of the molecules from the promoter DNA cancels any difference between them. 2.Structural differences between these complexes have been demonstrated. 3.At some promoters, a moribund complex is converted into a dead-end complex that still retains a short transcript but has no elongation activity. Therefore, the initiation pathway is branched into the conventional productive pathway and the abortive pathway that can lead towards a dead end. 4.The fates of a moribund complex are either inactivation as a dead-end complex, dissociation from the DNA, or direct conversion into a productive complex, and the rates of these reactions vary with the promoter. 5. There are factors that affect the fate of the moribund complex in a manner that depends on the promoter.
To examine the existence and significance of the branched pathway in vivo, we selected GreA and GreB for clues. At the lP_RAL promoter these factors enhance conversion of the moribund complex into the productive one, in the presence of high concentrations of initiating nucleoside triphosphate in vitro. If the branched mechanism exists in vivo, absence of the Gre factors should result in reduction of productive transcription from promoters at which the moribund complex is susceptible to these factors. We constructed a double-disruptant of E.coli, DgreA DgreB, and then arbitrarily selected 10 genes from among those whose levels of transcripts in the mutant strain were found to be lower than those in the parental greA^+greB^+ strain. Finally, the promoter for three of these genes, atpC (uncC), cspA, and rpsA, passed a further conventional test which confirmed that they displayed a branched initiation pathway in a reconstituted transcription system composed of purified components. The results obtained prove that the branched initiation pathway exists in vivo and is utilized in regulation of transcription initiation from some promoters, through modulation of the fraction of polymerase-promoter complexes entering each branch of the pathway. The determination of the level of GreB was observed to be constitutive. The level of GreA remained the same through the growth phase, and did not respond much to the richness of the culture media. However, it decreased into half in aerobic conditions, indicating that some genes are regulated by GreA. Therefore, the regulatory circuit responding to the levels of proteins involving GreA, namely the branched pathway mechanism, is working in cells. Less
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