Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays
Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays
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DOI：
10.1073/pnas.112318199
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发表时间：
2002-07-23
影响因子：
11.1
通讯作者：
Cohen, SN
中科院分区：
文献类型：
--
作者：
Bernstein, JA;Khodursky, AB;Cohen, SN
关于影响大肠杆菌中mRNA衰变的因素的信息，以及其他细菌中的推论，大部分是从对大约4,300种预测的大肠杆菌信息中不到25种的研究中收集的。为了更广泛地研究这些因素，我们使用双色荧光DNA微阵列以单基因分辨率检查了已知和预测的大肠杆菌mRNA的半衰期和稳态丰度。一种基于rRNA的微阵列数据的标准化策略被开发出来，以允许定量利福平转录阻滞后的mRNA衰减。我们发现，在全球范围内，mRNA半衰期在营养丰富的培养基和限定培养基中相似，其中世代时间大约是三倍。大肠杆菌的单个mRNA的稳定性范围很广，尽管大约80%的mRNA的半衰期在3到8分钟之间。通常观察到具有生物相关代谢功能的基因具有相似的稳定性。尽管有限数量的mRNA的半衰期与其丰度呈正相关，但我们发现，总体而言，mRNA稳定性的增加并不预示着丰度的增加。RNase E切割位点的密度也不高，RNase E被认为是启动E.大肠杆菌中，5'或3'非翻译区序列的折叠自由能也不能预测mRNA的半衰期。我们的研究结果确定了先前未被怀疑的mRNA衰变的功能在全球范围内，也表明，从个别基因转录的研究中得出的关于衰变的概括可能具有有限的适用性。
Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximate to4,300 predicted E coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E coli, although approximate to80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.