KH domains with impaired nucleic acid binding as a tool for functional analysis.

Abstract:

:In eukaryotes, RNA-binding proteins that contain multiple K homology (KH) domains play a key role in coordinating the different steps of RNA synthesis, metabolism and localization. Understanding how the different KH modules participate in the recognition of the RNA targets is necessary to dissect the way these proteins operate. We have designed a KH mutant with impaired RNA-binding capability for general use in exploring the role of individual KH domains in the combinatorial functional recognition of RNA targets. A double mutation in the hallmark GxxG loop (GxxG-to-GDDG) impairs nucleic acid binding without compromising the stability of the domain. We analysed the impact of the GDDG mutations in individual KH domains on the functional properties of KSRP as a prototype of multiple KH domain-containing proteins. We show how the GDDG mutant can be used to directly link biophysical information on the sequence specificity of the different KH domains of KSRP and their role in mRNA recognition and decay. This work defines a general molecular biology tool for the investigation of the function of individual KH domains in nucleic acid binding proteins.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Hollingworth D,Candel AM,Nicastro G,Martin SR,Briata P,Gherzi R,Ramos A

doi

10.1093/nar/gks368

subject

Has Abstract

pub_date

2012-08-01 00:00:00

pages

6873-86

issue

14

eissn

0305-1048

issn

1362-4962

pii

gks368

journal_volume

40

pub_type

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