Characterization the Functional RNA Polymerase Subunits of Xanthomonas campestris pv. campestris

• Main Authors: Ching-Yuan Cheng, 鄭景元
• Other Authors: Ming-Te Yang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/86020187733056970042

博士 === 國立中興大學 === 分子生物學研究所 === 97 === Prokaryotic RNA polymerase (RNAP) is a protein complex responsible for gene expression, consisting of α dimer, β, β′, and ω subunits. Initiation of gene transcription is allowed to proceed when a dissociable σ subunit associates with the core RNAP to form the holoenzyme, directing the holoenzyme to bind to the promoter upstream of the target gene. Functions of RNAP in E. coli have been well characterized, and the crystal structure analysis of Thermus aquaticus (Taq) RNAP has provided detail information about interactions among RNAP subunits. However, results of studies in AT-rich gram-positive bacteria, such as Bacillus subtilis, or Rhodobacter capsulatus with high G+C content showed that transcriptional regulation mechanisms of RNAPs of these bacteria are different from that of E. coli RNAP. Xanthomonas campestris pv. campestris (Xcc) has a G+C content of 65%, and the transcriptional mechanisms of this bacterium are not well-defined. The aim of this dissertation was to characterize the functions of RNAP subunits in Xcc, and results of this study were divided into three sections. In the first section, purification and characterization of Xcc RNAP with a His-tag fusion at either the C-terminus of α or β′ subunit were compared. Result showed that αHis-tag RNAP renders its binding to Ni-NTA stronger than β′His-tag RNAP. Further analyses demonstrated that αHis-tag core RNAP keeps the same ability to interact with σ factors. In addition, coexpression of Xcc RNAP subunits was carried out in E. coli with a plasmid bearing rpoC-rpoB-rpoAHis-tag genes, and in vivo reconstitution of Xcc RNAP subunits to form a core RNAP was successfully achieved. By using this approach, the purification of Xcc RNAP was simplified and the time-cost was reduced. The second section demonstrates the physiological roles of ω in Xcc. Expression of rpoZ was found to be closely related to the cell growth phase. Mutation in rpoZ had influence on expression of the downstream spoT gene. Additionally, the rpoZ mutant exhibited slower growth rate than that of the wild type, suggesting that rpoZ might be involved in regulating the stringent response of Xcc. Moreover, transcriptional activity of the recombinant Xcc core RNAP with ω coexpressed in E. coli had a 13-fold increase than that of the one lacking ω, indicating ω is an important element in maintenance the enzymatic activity of Xcc RNAP. The third section was focused on the expression and regulation of the ECF σ factor, RpoE (σE). RT-PCR analyses demonstrated that rpoE and rseA are in the same operon. The results of DNase I footprint analyses showed the binding by EσE on the σE-dependent upstream promoters of rpoE and rseA. The expression level of σE increases 20 ~ 40% in response to 42℃ or 5% EtOH stresses. Expression of heat-inducible σ32 decreased in rpoE mutant under heat shock, suggesting the role of σE in σ32 expression in response to heat shock.