High-throughput biochemical profiling reveals functional adaptation of a bacterial Argonaute

Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus the...

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Bibliographic Details
Published in:Molecular cell Vol. 82; no. 7; p. 1329
Main Authors: Ober-Reynolds, Benjamin, Becker, Winston R, Jouravleva, Karina, Jolly, Samson M, Zamore, Phillip D, Greenleaf, William J
Format: Journal Article
Language:English
Published: United States 07.04.2022
Subjects:
Argonaute Proteins - metabolism
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
DNA - genetics
Endonucleases - metabolism
Thermus thermophilus - genetics
Thermus thermophilus
single-molecule biophysics
siDNA
Argonaute
miRNA
high-throughput biochemistry
pAGO
siRNA
RNA silencing
TtAgo
ISSN:1097-4164, 1097-4164
Online Access:Get more information
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Summary:Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales.
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ISSN:1097-4164
1097-4164
DOI:10.1016/j.molcel.2022.02.026