Mtr4 RNA Helicase
Elizabeth Abrash '17 and Ulises Arbelo '16


I. Introduction

The Saccharomyces cerivisiae mRNA transport protein (Mtr4) is a RNA helicase that interacts with the RNA exosome. The exosome core is responsible for processing and degrading RNA.  In vivo function of the core requires several cofactors, on of which is Mtr4.  The RNA exosome core is conserved in eukaryotes and Archaea. In both cases the central channel of the exosome is known to be 8-10A wide, or wide enough to process single-stranded RNA. As an RNA helicase, Mtr4 is, in part, responsible for unwinding double stranded mRNA, so that it can be degraded.  

Mtr4 can act alone to unwind RNA, or it can interact with the Tr4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complexes. This complex consists of a poly(A)polymerase (Tr4/5) and a Zn-knuckle RNA-binding protein (Air1/2). In order to act on its own, Mtr4 requires a 3' overhang of ~5-6 nt. If the RNA substrate does not have this overhang, then it first binds the TRAMP complex until there is a minimal binding site for Mtr4.nbsp;

II. General Structure

Mtr4 includes a DExH core formed by two RecA domains, the N terminal beta hairpin, the Winged Helix domain, and the helical bundle domain. The , RecA-1 and RecA-2, bind RNA and ADP. The , packs with the beta strands of RecA-1 and latches on to RecA-2. RecA-2 is connected to the , by 15 residues and the WH domain packs against the . Mtr4 also features the in between the wings of the winged helix. The first two alpha helices of this insert are roughly perpendicular to each other. The overall structure of the dimer is assymetric; one subunit adopts a "closed" conformation in which the amino- and carboxy-termini are closer together than in the more "open" subunit. Each subunit is composed of two distinct domains connected by a hinge region. The N-terminal domain is responsible for dimerization and cAMP binding. The carboxy-terminal domain contains a helix-turn helix DNA binding motif, and is also responsible for DNA bending. 

III. cAMP Binding

An important recognition site for cAMP within CAP is the ionic bond formed between the side chain of Arg-82 and the negatively charged phosphate group of cAMP. In the crystal structure, the two cAMP molecules are buried deep within the beta roll and the C-helix. It is unclear how cAMP enters or leaves the binding site, but this probably requires the separation of the two subunits of the dimer, or the movement of the beta roll and the C helix away from each other. Other side-chain interactions between the protein and cAMP are hydrogen bonds occuring at Thr-127, Ser-128, Ser-83, and Glu-72. Additional hydrogen bonding between is seen between cAMP and the polypeptide backbone at residues 83 and 71

IV. DNA Binding

Once CAP has bound cAMP, it is ready to bind to the DNA. Binding occurs at the conserved sequence of 5'-AAATGTAGATCACATTT-3' Hydrogen bonds between the protein and the DNA phsophates occur at the backbone amide of residue 139, and the side chains of Thr-140, Ser-179, and Thr-182 In addition to these phosphate interactions, the side chains of Glu-181 and Arg-185, both emanating from the recognition helix directly contact the bases within the major groove of the DNA. Because of the way that the protein binds to the DNA, a kink of ~40 degrees occurs between nucleotide base pairs six and seven on each side of the dyad axis, 5'-TG-3' This sequence has been shown to favor DNA flexibility and bending in other systems as well. Because of this kink, an additional five ionic interactions and four hydrogen bonds are able to occur between the protein and the DNA strand. Examples of these new interactions occur between Lys-26, Lys-166, His-199 and the DNA sugar-phosphate backbone The DNA bend is integral to the mechanism of transcription activation. Not only does it place CAP in the proper orientation for interaction with RNA polymerase, but wrapping the DNA around the protein may result in direct contacts between upstream DNA and RNA polymerase. 

V. Activating Regions

Transcription activation by CAP requires more than merely the binding of cAMP and binding and bending of DNA. CAP contains an "activating region" that has been proposed to participate in direct protein-protein interactions with RNA polymerase and/or other basal transcription factors. Specifically, amino acids 156, 158, 159, and 162 have been proposed to be critical for transcription activation by CAP. These amino acids are part of a surface loop composed of residues 152-166 Researchers have concluded that the third and final step in transcription activation is this direct protein-protein contact between amino acids 156-162 of CAP, and RNA polymerase.

VI. References

Gunasekera, Angelo, Yon W. Ebright, and Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of the Escherichia coli Catabolite Gene Activator Protein. The Journal of Biological Chemistry 267:14713-14720.

Schultz, Steve C., George C. Shields, and Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex: The DNA Is Bent by 90 degrees Science 253: 1001-1007.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

Weber, Irene T., Gary L. Gilliland, James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic AMP-independent Mutant of Catabolite Activator Protein. The Journal of Biological Chemistry 262:5630-5636.

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

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