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Crystal structure and functional analysis of DEAD-box protein Dhh1p.

ABSTRACT: The control of mRNA translation and degradation are critical for proper gene expression. A key regulator of both translation and degradation is Dhh1p, which is a DEAD-box protein, and functions both to repress translation and enhance decapping. We describe the crystal structure of the N- and C-terminal truncated Dhh1p (tDhh1p) determined at 2.1 A resolution. This reveals that, like other DEAD-box proteins, tDhh1p contains two RecA-like domains, although with a unique arrangement. In contrast to eIF4A and mjDEAD, in which no motif interactions exist, in Dhh1p, motif V interacts with motif I and the Q-motif, thereby linking the two domains together. Electrostatic potential mapping combined with mutagenesis reveals that motifs I, V, and VI are involved in RNA binding. In addition, trypsin digestion of tDhh1p suggests that ATP binding enhances an RNA-induced conformational change. Interestingly, some mutations located in the conserved motifs and at the interface between the two Dhh1 domains confer dominant negative phenotypes in vivo and disrupt the conformational switch in vitro. This suggests that this conformational change is required in Dhh1 function and identifies key residues involved in that transition.

SUBMITTER: Cheng Z

PROVIDER: S-EPMC1370809 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Crystal structure and functional analysis of DEAD-box protein Dhh1p.

Cheng Zhihong Z Coller Jeff J Parker Roy R Song Haiwei H

RNA (New York, N.Y.) 20050629 8

The control of mRNA translation and degradation are critical for proper gene expression. A key regulator of both translation and degradation is Dhh1p, which is a DEAD-box protein, and functions both to repress translation and enhance decapping. We describe the crystal structure of the N- and C-terminal truncated Dhh1p (tDhh1p) determined at 2.1 A resolution. This reveals that, like other DEAD-box proteins, tDhh1p contains two RecA-like domains, although with a unique arrangement. In contrast to ...[more]

PMID: 15987810

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Project description:DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context of current models of Mss116p-facilitated splicing.

Dataset Information

Crystal structure and functional analysis of DEAD-box protein Dhh1p.

Publications

Crystal structure and functional analysis of DEAD-box protein Dhh1p.

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