Kinetics of Paused Ribosome Recycling in Escherichia coli
Kinetics of Paused Ribosome Recycling in Escherichia coli
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DOI：
10.1016/j.jmb.2009.09.020
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发表时间：
2009-11-27
影响因子：
5.6
通讯作者：
Hayes, Christopher S.
中科院分区：
文献类型：
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作者：
Janssen, Brian D.;Hayes, Christopher S.
细菌tmRNA-SmpB系统在称为“核糖体拯救”的过程中消除停滞的翻译复合物。tmRNA-SmpB特异性识别在截短的mRNA的3'端处或附近暂停的核糖体;因此，在可以从全长转录物中拯救暂停的核糖体之前，需要进行溶核mRNA加工。在这里，我们研究了核糖体的回收暂停全长和截短的mRNA。识别对应于每个暂停翻译复合物的肽基-tRNA，并使用其周转动力学来估计暂停核糖体在体内的半衰期。核糖体暂停在终止密码子的全长mRNA使用新生肽基序，干扰翻译终止和eliminantmRNA-SmpB活性。在tmRNA（+）细胞中（T-1/2=22 +/- 2.2 s），这些终止暂停的核糖体的肽酰-tRNA周转比在Delta tmRNA细胞中（T-1/2=32 +/- 1.6 s）稍快。释放因子（RF）1的过表达大大加速了tmRNA（+）和Δ tmLRNA细胞中终止暂停核糖体的肽酰-tRNA周转，而其他终止因子对再循环几乎没有影响。与低效的翻译终止相反，核糖体从缺少框内终止密码子的截短转录物的再循环被tmRNA-SmpB显著加速。然而，在Delta tmRNA细胞中，肽基-tRNA仍然以显著的速率（t（1/2）=61 +/- 7.3 s）从非停止-暂停的核糖体转换。RF-1，RF-3和核糖体再循环因子在Delta tmRNA细胞中的过表达未能加速核糖体从不间断mRNA的再循环。这些结果表明，tmRNA-SmpB活性是由mRNA切割速率限制，RF-3和核糖体再循环因子不构成一个tmRNA-independent救援途径，如前所述。Delta tmRNA细胞中来自非停止暂停核糖体的肽基-tRNA周转表明存在另一种未表征的核糖体拯救途径。(C)2009爱思唯尔有限公司保留所有权利。
The bacterial tmRNA-SmpB system recycles stalled translation complexes in a process termed 'ribosome rescue.' tmRNA-SmpB specifically recognizes ribosomes that are paused at or near the 3' end of truncated mRNA; therefore, nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics was used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA-SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA(+) cells (T-1/2=22 +/- 2.2 s) than in Delta tmRNA cells (T-1/2=32 +/- 1.6 s). Overexpression of release factor (RF) 1 greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA(+) and Delta tmLRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA-SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t(1/2)=61 +/- 7.3 s) in Delta tmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor in Delta tmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA-SmpB activity is rate limited by mRNA cleavage, and that RF-3 and ribosome recycling factor do not constitute a tmRNA-independent rescue pathway, as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in Delta tmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway. (C) 2009 Elsevier Ltd. All rights reserved.