Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNA_i ^Met Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro

Authors

Yan Li, Agricultural University of Hebei
Joseph Burclaff, Washington University in St. Louis
James T. Anderson, Marquette UniversityFollow

Document Type

Article

Language

eng

Publication Date

1-28-2016

Publisher

Public Library of Science

Source Publication

PLoS One

Source ISSN

1932-6203

Original Item ID

DOI: 10.1371/journal.pone.0148090

Abstract

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNA_i^Met. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNA_i^Met turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNA_i^Met turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

Comments

Published version. PLos One, Vol. 11, No. 1 (January 28, 2016): e0148090. DOI. © 2016 Public Library of Science (PLoS). Used with permission.

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Li, Yan; Burclaff, Joseph; and Anderson, James T., "Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNA_i ^Met Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro" (2016). Biological Sciences Faculty Research and Publications. 501.
https://epublications.marquette.edu/bio_fac/501