Funk C J, Harwood S H, Rohrmann G F
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA.
Virology. 1998 Feb 1;241(1):131-40. doi: 10.1006/viro.1997.8961.
An in vitro system for baculovirus late transcription was developed that allows comparison of conditions that affect transcription initiation and elongation. A series of synthetic promoters was constructed based on the baculovirus late p6.9 promoter. The modified promoters were designed with a cytidine-free region downstream of the late promoter in order to yield paused transcripts of defined lengths in the absence of CTP. Transcription was found to be more efficient from a supercoiled template than from a linear template for this promoter. The stalled transcription complex remained competent and could be elongated in the presence of a full set of nucleotides. This made it possible to separately test the effects of heat treatment and inhibition by sarkosyl and heparin on initiation and elongation. Elongation complexes were more resistant than initiation complexes to each of these treatments. Furthermore a 1-2 mM MgCl2 concentration is critical for optimal initiation, but elongation can proceed in the presence of MgCl2 concentrations as high as 20 mM.
开发了一种用于杆状病毒晚期转录的体外系统，该系统可比较影响转录起始和延伸的条件。基于杆状病毒晚期p6.9启动子构建了一系列合成启动子。修饰后的启动子在晚期启动子下游设计了一个无胞苷区域，以便在没有CTP的情况下产生确定长度的暂停转录本。发现对于该启动子，超螺旋模板的转录比线性模板更有效。停滞的转录复合物仍然具有活性，并且在全套核苷酸存在的情况下可以延伸。这使得分别测试热处理以及十二烷基肌氨酸钠和肝素抑制对起始和延伸的影响成为可能。延伸复合物比起始复合物对这些处理中的每一种都更具抗性。此外，1-2 mM的MgCl2浓度对于最佳起始至关重要，但在高达20 mM的MgCl2浓度下延伸仍可进行。
