Gene expression analysis via quantitative measurement of biomolecules in Escherichia coli

Abstract

To obtain an exact model of gene expression is important for control of cellular metabolism and a target for antibiotics. A bacterial organism Escherichia coli (E. coli) has been used as a simple model system. Gene expression is a series of chemical reaction occurring between biomolecules in cell. Therefore, the numbers and their unit used to represent gene expression are chemical rate constants. Recently, scientists are interested in how the numbers are changed by small molecules or proteins. But, there is always a limitation for the observation of gene expression in cell because it is impossible to see native proteins in cell and their interactions with other proteins. In this thesis, quantitative measurement of messenger RNAs (mRNAs) and proteins were used to describe transcriptional dynamics in E. coli. The abundant of mRNAs that contain a specific sequence in a clue for the transcription elongation of RNAP. And visualization of fluorescent protein tagged RNAP gives the time required to transcribe a gene. Firstly, I report a physical analysis of transcription time in the expression of lacZ gene in E. coli. I proved that an antibiotics targeting rho factor (bicyclomycin) is a key molecule to separate E. coli RNAP and ribosome during active transcription and translation. Transcription time equation analysis enables us to obtain transcription initiation dynamics and elongation dynamics simultaneously. Secondly, I report the dynamical re-location of E. coli chromosome in cell during active transcription and translation. I measured the cellular location of the gene by fluorescent protein (FP) tagged DNA binding protein, and actively transcribing RNAP by FP tagged T7 RNAP. Then I obtained dynamical information such as transcriptional on rate and transcription elongation rate. I also found that the fluorescent loci of gene being transcribed moves toward the cytoplasm from nucleoid, and translation enhances the re-location of gene. Lastly, I compared FPs tagged to DNA binding protein in cells to obtain better fluorescent images from experiments. I used two factors represent DNA binding activity of FP tagged tet repressors (TetR-FP) then compared. As a result, I found mCrimson3 and EYFP are good for tagging on DNA binding protein for the observation of fluorescent focus in cell. Among them, mCrimson3 is an exceptional one (probably one the best).