Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, New York, USA.
J Bacteriol. 2012 Nov;194(22):6233-9. doi: 10.1128/JB.01223-12. Epub 2012 Sep 14.
Many Escherichia coli mRNAs are degraded by a 5'-end-dependent mechanism in which RppH-catalyzed conversion of the 5'-terminal triphosphate to a monophosphate triggers rapid endonucleolytic cleavage by RNase E. However, little is understood about what governs the decay rates of these transcripts. We investigated the decay of three such messages--rpsT P1, yfcZ, and ydfG--to characterize the rate-determining step in their degradation. The steady-state ratio of monophosphorylated to triphosphorylated rpsT P1 and yfcZ mRNA indicates that their decay rate is limited by cleavage of the monophosphorylated intermediate, making RNase E critical for their rapid turnover. Conversely, the decay rate of ydfG is limited by generation of the monophosphorylated intermediate; therefore, either RNase E or its less abundant paralog RNase G is sufficient for rapid ydfG degradation. Although all three transcripts are stabilized when RppH is absent, overproducing RppH does not accelerate their decay, nor does RppH overproduction appear to influence the longevity of most other messages that it targets. The failure of excess RppH to hasten rpsT P1 and yfcZ degradation despite increasing the percentage of each that is monophosphorylated is consistent with the observation that pyrophosphate removal is not the rate-limiting step in their decay. In contrast, neither the ydfG decay rate nor the fraction of ydfG transcripts that are monophosphorylated increases when the cellular concentration of RppH is raised, suggesting that, for some RppH targets, the rate of formation of the monophosphorylated intermediate is limited by an ancillary factor or by a step that precedes pyrophosphate removal.
许多大肠杆菌 mRNA 是通过 5'-端依赖机制降解的，其中 RppH 催化的 5'-末端三磷酸转化为单磷酸触发 RNase E 的快速内切核酸酶切割。然而，对于这些转录物的降解速率由什么决定，人们知之甚少。我们研究了三种这样的消息--rpsT P1、yfcZ 和 ydfG--的衰变，以表征它们降解的速率决定步骤。稳态的 rpsT P1 和 yfcZ mRNA 的单磷酸化与三磷酸化之比表明，它们的衰变速率受单磷酸化中间产物的切割限制，这使得 RNase E 对其快速周转至关重要。相反，ydfG 的衰变速率受单磷酸化中间产物的生成限制；因此，RNase E 或其丰度较低的同工酶 RNase G 足以快速降解 ydfG。尽管当 RppH 不存在时，所有三种转录本都稳定，但过量产生 RppH 不会加速它们的衰变，过量产生 RppH 似乎也不会影响它针对的大多数其他消息的寿命。尽管过量的 RppH 增加了每个 rpsT P1 和 yfcZ 的单磷酸化比例，但并没有加速它们的降解，这与观察到焦磷酸的去除不是它们衰变的限速步骤是一致的。相比之下，当 RppH 的细胞浓度升高时，ydfG 的衰变速率或 ydfG 转录本中单磷酸化的比例都不会增加，这表明对于某些 RppH 靶标，单磷酸化中间产物的形成速率受到辅助因子或焦磷酸去除之前的步骤的限制。
