噬菌體Xp10基因轉錄的控制

Abstract

Xp10?水稻白葉枯病病原菌Xanthomonas campestris pv. oryzae之溶解性噬菌體，當Xp10感染寄主細菌後，菌體內之轉錄系統由對rifampicin敏感轉變?對rifampicin有抗性，經酵素分析後，發現寄主原本對rifampicin敏感的RNA聚合?活性，因Xp10的感染而下降，接著又產生一抗rifampicin的RNA聚合?，取代寄主的RNA聚合?。 ?了探討寄主RNA聚合?對Xp10基因體轉錄的情形及其活性如何因Xp10感染而下降，因此分別以PEG沈澱法、DEAE-cellulose, heparin sepharose 4B及blue dextran sepharose 4B等層析法將感染前、後之寄主RNA聚合?加以純化，經7.5％ SDS-polyacrylamide膠體電泳分析，發現寄主RNA聚合?次單位中α2ββ'σ中的σ次單位因Xp10感染而遺失；同時感染後之寄主RNA聚合?的模版專一性也因而改變，未感染者可利用Xp10 DNA及寄主DNA?模版合成RNA，而感染後者則不能轉錄Xp10 DNA及寄主DNA，僅能轉錄poly d(A－T)。接著再比較其差異性，譬如對於spermidine的促進作用，actinomycin D及blue dextran之抑制作用亦有所差異，其他特性如RNA合成之最適二價陽離子濃度、最適溫度、最適pH?及對於鹽類、NEM、rifampicin、congo red、heparin、ethidium bromide之抑制作用並沒有顯著改變；以polyacrylamide-agarose混合膠體電泳分析兩種RNA聚合?對Xp10 DNA之轉錄產物，得知感染前之寄主RNA聚合?可得到五條主要的RNA帶，而感染後之寄主RNA聚合?僅得到較弱的兩條RNA帶，?了繼續追蹤這兩種酵素對Xp10 DNA轉錄的專一性是否改變，因此分別以其轉錄Xp10 DNA所得之32P-RNA?偵測體(probe)，對Xp10 DNA片斷進行DNA-RNA雜交試驗，發現兩者均只能轉錄Xp10 DNA左端25?30％部份。接著以從Xp10感染菌體中分離出的Xp10抑制因數，對感染前之寄主RNA聚合?作用，發現可抑制其對Xp10 DNA及寄主DNA之轉錄，而與感染後之寄主RNA聚合?特性相似。但是此抑制因數不改變感染後寄主RNA聚合?對Xp10 DNA及寄主DNA之轉錄能力。 Xp10感染後，除了寄主RNA聚合?被抑制外，又產生一Xp10 RNA聚合?負責轉錄Xp10晚期基因；同樣以上述純化步驟加以純化，經7.5％ SDS膠體電泳分析後，得知它?分子量98,000之單一蛋白質RNA聚合?，嗜好以加熱處理過之Xp10 DNA、calf thymusDNA及poly d(A－T)?模版合成RNA，對自然、雙股(native, double strand)的DNA及poly d(C)•poly d (G)之轉錄能力則較差，但只合成poly r(G)，不合成poly r(C)。若以加熱處理之calf thymus DNA?模版，受質ATP單獨存在時，可合成poly r(A)。正常之轉錄作用須有四種受質、DNA模版及鎂離子同時存在；RNA合成之最適鎂離子濃度?16 mM，最適溫度?37℃，最適pH??8.0，受質ATP之Km??26μm，spermidine可抑制其酵素活性，50mM的KCl可抑制其90％的酵素活性。60μg/ml的actinomycin D, heparin, blue dextran, ethidium bromide可完全抑制其活性，而對同一濃度的rifampicin及streptovaricin具有抗性。其酵素活性部位(active site)不具－SH基。Xp10 RNA聚合?轉錄Xp10 DNA之RNA產物，經polyacrylamide-agarose混合膠體分析後，得到兩條主要的RNA帶，再以此32P-RNA?偵測體，對Xp10 DNA片斷做DNA-RNA雜交分析，得知Xp10 RNA聚合?僅轉錄Xp10 DNA右端70?75％部份。 ?了驗證在生體內(in vivo) Xp10基因體轉錄的方向，是否亦由Xp10 DNA物理圖譜左端進行，因此分別抽取Xp10感染後不同時間菌體內之32P-RNA，再依上法做雜交試驗，結果證明仍由左端向右端的方向轉錄。 由以上結果歸納如下：Xp10感染寄主細菌後，寄主RNA聚合?負責轉錄Xp10早期基因，再由早期基因產物－Xp10抑制因數抑制寄主RNA聚合?活性，再以另一早期基因產物－Xp10 RNA聚合?負責轉錄Xp10晚期基因。

Xp10 is a virulent phage of Xanthomonas campestris pv. onyzae. When the host bacteria were infected with Xp10, the activity of rifampicin sensitive transcription, originally presents in the host, was dramatically decreased, meanwhile the rifampicin resistant transcription was induced. Both the RNA polymerases from uninfected and infected cells were purified to homogeneity by PEG precipitation, DEAE-cellulose, heparin sepharose 4B and blue dextran sepharose 4B chromatography. Properties of these two enzymes were compared. When the protein subunits of both enzymes were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis, the losing of σ subunit from the infected host RNA polymerase was observed. In the in vitro assay systems, the infected-host RNA polymerase was unable to transcribe native DNA efficiently, while the uninfected-host RNA polymerase was able to transcribe native DNA. The optimal concentration of divalent cation, temperature and pH for both enzyme activities were very similar. The effects of salts and some reagents, such as rifampicin, congo red, heparin, N-ethylmaleimide, ethidium bromide on both enzymes were also similar. There were some differences between the effects of spermidine, actinomycin D and blue dextran. The transcriptional products of both enzymes upon Xp10 DNA were analyzed by polyacrylamide-agarose composite gel electrophoresis and autoradiography. Five major radioactive RNA bands were visualized in the products of uninfected-host RNA polymerase and two major radioactive RNA bands were visualized in the products of infected-host RNA polymerase. The transcribed regions on Xp10 DNA by both enzymes were analyzed by DNA-RNA hybridization, both enzymes could only transcribe leftmost 25-30% of the total Xp10 genome, although the transcriptional efficiency of infected-host RNA polymerase was less than that of uninfected-host RNA polymerase. An inhibitor, which is protein in nature, had been isolated from Xp10 infected cells. It could inhibit the transcription of uninfected-host RNA polymerase upon native DNA, but not upon poly d(A-T). The Xp10 induced RNA polymerase was also purified to homogeneity by PEG preciptation, DEAE-cellulose, heparin sepharose 4B and blue dextran sepharose 4B chromatography. It possesses a single polypeptide chain with a molecular weight of 98,000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA and poly d(A-T) as templates for in vitro RNA synthesis. The optimal concentration of divalent cation (16mM), temperature(37℃) and pH(8.0) for RNA synthesis were determined. 90% of the enzyme activity was inhibited by the presence of 0.05 M KC1. The enzyme activity was inhibited by spermidine which could enhance the activity of host RNA polymerase. The four substrates (ATP, CTP, GTP, UTP,) were essential for RNA synthesis. The Km of ATP was 26μM. Poly r(A) could be formed when denatured calf thymus DNA was used as a template in the absence of CTP, GTP and UTP. The enzyme activity did not alter by the presence of N-ethylmaleimide, it suggested that -SH group was not required for the active site of Xp10 RNA polymerase. The enzyme activity was blocked by actinomycin D, heparin, blue dextran, ethidium bromide, but it was resistant to rifampicin and streptovaricin. The in vitro transcriptional products of Xp10 DNA by using Xp10-induced RNA polymerase were analyzed by polyacrylamide-agarose composite gel electrophoresis and autoradiograph. Two major radioactive RNA bands were visualized. The transcribed regions on Xp10 DNA were also analyzed by DNA-RNA hybridization. It was found that Xp10-induced RNA polymerase could only transcribe the rightmost 70-75% of the total Xp10 genome. The results of in vivo transcriptional experiments show that the Xp10 genome was transcribed in the same direction as in vitro. It was concluded that Xp10 early gene were firstly transcribed by host RNA polymerase. Among the early gene products, one is an inhibitor inhibiting the host RNA polymerase, and the other is a new RNA polymerase taking responsibility of the transcription of Xp10 late genes.