|Budget Amount *help
¥14,600,000 (Direct Cost: ¥14,600,000)
Fiscal Year 2002: ¥9,100,000 (Direct Cost: ¥9,100,000)
Fiscal Year 2001: ¥5,500,000 (Direct Cost: ¥5,500,000)
Global regulation of transcription of total genes on the genome is one of the research front after completion of the genome sequencing. A decade ago we found that the total number of RNA polymerase molecules (about 2000) is less than the total number of genes (about 4000) on the E. coli genome. Based on this finding, we proposed that the RNA polymerase chooses the genes to transcribe under a given environmental condition, and the modulation of specificity and activity of RNA polymerase plays a major role in switching transcription pattern upon expose to different conditions. This research project has been undertaken to reveal this model. Followings are the major results obtained in this study:
1)E. coli RNA polymerase is functionally differentiated into various forms of transcription apparatus in two steps. In the first-step, the core enzyme associates with one of seven molecular species of the sigma subunit. We have determined both the intracellular concentrations of all seven sigma su
bunits under various culture conditions and the binding affinity of each sigma subunit to the core enzyme. From these two values, we succeeded to estimate the intracellular concentration of seven holoenzymes, each carrying one specific sigma factor.
2) Transcription patterns were analyzed, using ONA chips, for E coli mutants, each lacking one sigma factor. Based on the transcriptome analysis, we estimated the genes under the control of each sigma subunit.
3) RNA polymerase holoenzyme is further differentiated by interaction with one (or two in a few cases) of a total 266 transcription factors. For identification of genes under the control of each transcription factor, transcriptome and proteome analyses have been carried out for E coli mutants, each lacking one specific transcription factor.
4) To examine the prediction for transcription factor functions, in vitro transcription assay of the candidate promoters is being carried out using purified RNA polymerase and transcription factors.
5) Specific antibodies have been made against purified transcription factors and the determination of the intracellular concentrations of each transcription factor is in progress.
6) Fractionation method of stationary-phase cultures of E. coli into homogenous cell populations has been established by using Percoll gradient centrifugation. After analyses of changes in gene expression pattern and of transcription apparatus for fractionated cell populations, we propose a sequential differentiation model for the stationary-phase adaptation of E coli. Less