COUPLING OF RIBOSOMAL-RNA TRANSCRIPTION AND RIBOSOMAL ASSEMBLY INVIVO - FORMATION OF ACTIVE RIBOSOMAL-SUBUNITS IN ESCHERICHIA-COLI REQUIRES TRANSCRIPTION OF RIBOSOMAL-RNA GENES BY HOST RNA-POLYMERASE WHICH CANNOT BE REPLACED BY BACTERIOPHAGE-T7 RNA-POLYMERASE
COUPLING OF RIBOSOMAL-RNA TRANSCRIPTION AND RIBOSOMAL ASSEMBLY INVIVO - FORMATION OF ACTIVE RIBOSOMAL-SUBUNITS IN ESCHERICHIA-COLI REQUIRES TRANSCRIPTION OF RIBOSOMAL-RNA GENES BY HOST RNA-POLYMERASE WHICH CANNOT BE REPLACED BY BACTERIOPHAGE-T7 RNA-POLYMERASE
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DOI：
10.1006/jmbi.1993.1311
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发表时间：
1993-06-05
影响因子：
5.6
通讯作者：
NIERHAUS, KH
中科院分区：
文献类型：
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作者：
LEWICKI, BTU;MARGUS, T;NIERHAUS, KH
用T7RNA聚合酶或宿主RNA聚合酶作为转录酶，研究了活体中rrnBoperon的转录和相应的核糖体的形成。该质粒上的23个S核糖体核糖核酸基因携带A1067→T突变，使其对硫代链球菌耐药。通过扫描1067核苷酸周围的序列放射自显影图，定量了含有质粒携带的23 S rRNA与染色体携带的rRNA的颗粒比例，精度优于10%。当用噬菌体T7 RNA聚合酶转录质粒rnBoperon时，在诱导后期，高达80%的rRNA合成是由质粒控制的(脉冲标记)，其中大部分(约85%)被降解。细胞内积聚了50个S颗粒，携带有70个S核糖体中所没有的完整的核糖核酸，即携带有质粒携带的核糖核酸的颗粒没有进入活性核糖体池。含有质粒源rRNAs的颗粒在体外蛋白质合成中也几乎没有活性。重组分析表明，rRNA具有一定的功能。在不同表达水平的rrnB操纵子中发现了相同的模式，这表明不是过量生产或rRNA，而是T7转录酶对观察到的效果负责。然而，用宿主RNA聚合体转录rnB操纵子时，诱导后的生长不受影响，细胞内30 S：50 S：70 S的比例没有改变，50 S亚基和70 S核糖体都含有大量的质粒源rRNA，并且含有质粒源rRNA的颗粒在体外是活跃的。当T7RNA聚合酶在25℃而不是37℃下诱导rRNA转录时，观察到几乎正常的模式。失活的50个S颗粒没有聚集，在70个S核糖体的池中发现了大量的质粒携带的rRNA。降低诱导温度会降低T7RNA聚合酶的转录速度，在37℃时，T7RNA聚合酶的转录速度是宿主聚合酶的5倍。结果表明，活体活性核糖体亚单位的形成需要rRNAs转录速率和组装过程的良好适应，强调了rRNA转录和活体核糖体组装之间耦合的重要性。在37℃下，T7 RNA聚合酶不能取代宿主RNA聚合酶。
Transcription of a plasmid-locatedrrnBoperon and the corresponding formation of ribosomesin vivowere studied using either T7 RNA polymerase or host RNA polymerase as transcriptase. The 23 S rRNA gene on the plasmid carried an A1067 → T mutation, which confers resistance against the drug thiostrepton. The proportion of particles containing plasmid-borne 23 S rRNAversuschromosome-borne rRNA was quantified with a precision of better than 10% by scanning sequence autoradiograms around nucleotide 1067. The activity of these particles was determined in the presence of thiostrepton which exclusively abolishes the activity of chromosomal wild-type ribosomes.When the plasmidrrnBoperon was transcribed with phage T7 RNA polymerase, up to 80% of the rRNA synthesis was plasmid-directed (pulse labelling) in the late induction phase, most of which (about 85%) became degraded. The cells accumulated 50 S particles with plasmid-borne intact rRNA that was hardly found in 70 S ribosomes, i.e. particles harbouring plasmid-borne rRNA did not enter the pool of active ribosomes. The particles with plasmid-derived rRNAs were also practically inactive in protein synthesisin vitro. However, the rRNA was functional as shown by reconstitution analysis. The same patterns were found at various expression levels of the plasmid rrnB operon, indicating that not the overproduction or rRNA but rather the T7 transcriptase was responsible for the observed effects. However, when the plasmid rrnB operon was transcribed with host RNA polymerse, growth was not affected upon induction, the 30 S to 50 S to 70 S ratios in the cell were not altered, both 50 S subunits and 70 S ribosomes contained large amounts of plasmid-borne rRNA, and the particles with plasmid-derived rRNA were active in vitro.When the induction of rRNA transcription by T7 RNA polymerase was performed at 25°C instead of 37°C, an almost normal pattern was observed. Inactive 50 S particles did not accumulate, and large amounts of plasmid-borne rRNA were found in the pool of 70 S ribosomes. Lowering the induction temperature reduces the transcription rate by T7 RNA polymerase, which is five times faster at 37°C than the host polymerase. The results suggest that the formation of active ribosomal subunitsin vivorequires a fine adaptation of the transcription rate of rRNAs and the assembly process, underlining the importance of a coupling between rRNA transcription and ribosome assemblyin vivo.T7 RNA polymerase cannot replace the host RNA polymerase in this process at 37°C.