2016
DOI: 10.1093/nar/gkw062
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Abstract: The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and preve… Show more

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Cited by 19 publications
(18 citation statements)
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“…An in vitro transcribed 30mer RNA containing a single 4-thiouridine at position 15 was labeled with iodoacetamido fluorescein and used for fluorescence anisotropy measurements (9,34). Activity assays were performed with a 21mer RNA under multiple turnover conditions (35). Reaction mixtures were analyzed by anion exchange HPLC and educt and product of the reaction were quantified based on the absorption at 260 nm.…”
Section: Methodsmentioning
confidence: 99%
“…An in vitro transcribed 30mer RNA containing a single 4-thiouridine at position 15 was labeled with iodoacetamido fluorescein and used for fluorescence anisotropy measurements (9,34). Activity assays were performed with a 21mer RNA under multiple turnover conditions (35). Reaction mixtures were analyzed by anion exchange HPLC and educt and product of the reaction were quantified based on the absorption at 260 nm.…”
Section: Methodsmentioning
confidence: 99%
“…RNA was then fluorescein labeled by incubating the RNA at a concentration between 30 and 100 µM in the presence of 10 mM fluorescein in 100 mM sodium phosphate buffer (pH 8.0) for 24 h at room temperature in the dark (Ramos and Varani 1998;Audin et al 2016). Afterward, the RNA was purified by three subsequent rounds of RNA precipitation.…”
Section: Fluorescein-labeling Of Rnamentioning
confidence: 99%
“…Could this difference account for the intrinsic distributive activity of the plant Exo9 while bacterial PNPases and archaeal exosomes are processive enzymes? In fact, the processivity of these prokaryotic exoribonucleases is primarily determined by the RNA binding affinity at the entrance of the channel 6 , 40 , 41 . Mutations that decrease RNA binding affinity at the channel entrance are sufficient to switch a processive S. solfataricus exosome into a distributive enzyme 41 .…”
Section: Discussionmentioning
confidence: 99%
“…In fact, the processivity of these prokaryotic exoribonucleases is primarily determined by the RNA binding affinity at the entrance of the channel 6 , 40 , 41 . Mutations that decrease RNA binding affinity at the channel entrance are sufficient to switch a processive S. solfataricus exosome into a distributive enzyme 41 . Albeit all three active sites inside the channel contribute to degradation, the RNA substrate moves faster between active sites than the cleavage rate.…”
Section: Discussionmentioning
confidence: 99%
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