# EUROISMAR 2019

25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

## Structure and Dynamics of Exoribonucleases

Not scheduled
4h
Harnack House and Henry Ford Building

Board: 347
Poster

Jan Overbeck

### Description

Xrn1 and Xrn2 are the main 5’-exoribonucleases in the cytoplasm, respectively the nucleus. These enzymes play a major role in the degradation of long RNA molecules and have been subject to a number of functional and structural studies (Xiang 2009, Chang 2011, Jinek 2011, Tesina 2019). To fully understand the underlying molecular mechanism of these essential enzymes, we here solved the crystal structure of the Xrn2 protein core. The Xrn2 zinc finger that is located next to the active site of the core domain of the enzyme was not visible in the crystallographic data and was structurally characterized using NMR spectroscopy. We show that this zinc finger binds RNA in vitro and contributes to the overall RNA affinity of Xrn2. We use methyl TROSY NMR experiments on the 100 kDa protein core and used site directed mutagenesis to obtain a resonance assignment for most of the 48 Ile-$\delta$1 methyl groups. These methyl probes were then used to record 1H13C-MQ and 13C-SQ CPMG experiments that display extensive dynamics on the $\mu$s-ms timescale in a region of the protein that surrounds the active site. These methyl relaxation data were complemented and confirmed with 19F-based CPMG experiments that we recorded on a number of samples that contained site specific fluor labels. In addition, the methyl group assignments were used to probe the interaction of Xrn2 with substrate RNA and with its main activator Rai1. Overall, our results combine static structures from X-ray crystallography with the state-of-the-art NMR methodology to obtain detailed insights into enzyme function.

References
(1) Chang et al., Nat. Struct. Mol. Biol. 18, 270–276 (2011)
(2) Jinek et al., Mol. Cell 41, 600–608 (2011)
(3) Xiang et al., Nat. Lett. 458, 784-788 (2009)
(4) Tesina et al., Nat. Struct. Mol. Biol. 26, 275-280 (2019)

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