Bogan J A, Helmstetter C E
Department of Biological Sciences, Florida Institute of Technology, Melbourne, 32901, USA.
J Bacteriol. 1996 Jun;178(11):3201-6. doi: 10.1128/jb.178.11.3201-3206.1996.
The potential role of mioC transcription as a negative regulator of initiation of chromosome replication in Escherichia coli was evaluated. When initiation was aligned by a shift of dnaC2(Ts) mutants to nonpermissive temperature (40 degrees C), mioC transcript levels measured at the 5' end or reading through oriC disappeared within one mass doubling. Upon return to permissive temperature (30 degrees C), the transcripts reappeared coordinately about 15 min after the first synchronized initiation and then declined sharply again 10 min later, just before the second initiation. Although these observations were consistent with the idea that mioC transcription might have to be terminated prior to initiation, it was found that the interval between initiations at permissive temperature, i.e., the eclipse period, was not influenced by the time required to shut down mioC transcription, since the eclipse was the same for chromosomes and minichromosomes which lacked mioC transcription. This finding did not, in itself, rule out the possibility that mioC transcription must be terminated prior to initiation of replication, since it might normally be shut off before initiation, and never be limiting, even during the eclipse. Therefore, experiments were performed to determine whether the continued presence of mioC transcription during the process of initiation altered the timing of initiation. It was found that minichromosomes possessing a deletion in the DnaA box upstream of the promoter transcribed mioC continuously and replicated with the same timing as those that either shut down expression prior to initiation or lacked expression entirely. It was further shown that mioC transcription was present throughout the induction of initiation by addition of chloramphenicol to a dnaA5(Ts) mutant growing at a semipermissive temperature. Thus, transcription through oriC emanating from the mioC gene promoter is normally inhibited prior to initiation of replication by the binding of DnaA protein, but replication can initiate with the proper timing even when transcription is not shut down; i.e., mioC does not serve as a negative regulator of initiation. It is proposed, however, that the reappearance and subsequent disappearance of mioC transcription during a 10-min interval at the end of the eclipse serves as an index of the minimum time required for the establishment of active protein-DNA complexes at the DnaA boxes in the fully methylated origin region of the chromosome. On this basis, the eclipse constitutes the time for methylation of the newly formed DNA strands (15 to 20 min at 30 degrees C) followed by the time for DnaA protein to bind and activate oriC for replication (10 min).
评估了mioC转录作为大肠杆菌染色体复制起始负调控因子的潜在作用。当通过将dnaC2(Ts)突变体转移到非允许温度（40℃）来使复制起始同步时，在5'端测量的或通读oriC的mioC转录本水平在一个群体倍增时间内消失。回到允许温度（30℃）后，转录本在第一次同步起始后约15分钟协同重新出现，然后在10分钟后，即在第二次起始前再次急剧下降。尽管这些观察结果与mioC转录可能必须在起始前终止的观点一致，但发现允许温度下起始之间的间隔，即隐蔽期，不受关闭mioC转录所需时间的影响，因为对于缺乏mioC转录的染色体和微型染色体，隐蔽期是相同的。这一发现本身并不排除mioC转录必须在复制起始前终止的可能性，因为它可能通常在起始前就被关闭，并且即使在隐蔽期也从不成为限制因素。因此，进行了实验以确定在起始过程中mioC转录的持续存在是否会改变起始时间。发现具有启动子上游DnaA框缺失的微型染色体持续转录mioC，并与那些在起始前关闭表达或完全缺乏表达的微型染色体以相同的时间进行复制。进一步表明，在半允许温度下生长的dnaA5(Ts)突变体中加入氯霉素诱导起始的整个过程中都存在mioC转录。因此，源自mioC基因启动子的通过oriC的转录在复制起始前通常通过DnaA蛋白的结合而受到抑制，但即使转录不关闭，复制也能以适当的时间起始；即，mioC不作为起始的负调控因子。然而，有人提出，在隐蔽期末端的10分钟间隔内mioC转录的重新出现和随后的消失作为在染色体完全甲基化的起始区域的DnaA框处建立活性蛋白质-DNA复合物所需最短时间的指标。在此基础上，隐蔽期包括新形成的DNA链甲基化的时间（30℃下为15至20分钟），随后是DnaA蛋白结合并激活oriC进行复制的时间（10分钟）。
