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Molecular Mechanism of the TRAMP Complex

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2011, Doctor of Philosophy, Case Western Reserve University, Biochemistry.

The TRAMP complex (Trf4/Air2/Mtr4 polyadenylation) is required for quality control and 3’ maturation of many RNAs in the nucleus. The TRAMP complex consists of three units, the poly(A) polymerase Trf4/5p, the zinc-knuckle protein Air1/2p, and the Ski2-like RNA helicase Mtr4p. Using recombinant Saccharomyces cerevisiae TRAMP complex, I found that the RNA helicase Mtr4p modulates activities of the polymerase Trf4p and restricts the length of oligo(A) appended after addition of ~4 nt. The modulation does not require duplex unwinding but relies on sensing of oligo(A) length by Mtr4p. Trf4p, in turn, stimulates the unwinding activity of Mtr4p independent of polyadenylation. Polyadenylation of duplex RNAs by Trf4p creates a landing site for Mtr4p to start unwinding, the length of oligo(A) required coincides with restriction of adenylation by Mtr4p. The functional cross-talk between Trf4p and Mtr4p in the TRAMP complex promotes addition of a minimum oligo(A) tail (~4 nt) and may be critical for sorting of polyadenylated RNAs in vivo and for subsequent specific target processing by the nuclear exosome.

The S. cerevisiae DEAD-box protein Ded1p unwinds RNA duplexes by local strand separation instead of translocation. In this study, I established that distinct units of Ded1p exist on single-stranded and duplex regions; Ded1p unit(s) bound to single-stranded regions facilitates loading of additional unit(s) of Ded1p to duplex regions for unwinding. I further analyzed Ded1p binding to single-stranded RNA in the presence of analogs for different states of ATP hydrolysis. Each Ded1p monomer binds ~10 nt ssRNA in the presence of the non-hydrolyzable analog ADPNP. The ground state analog ADP-BeFx and the transition state analog ADP-AlF4 lead to binding of more Ded1p molecules to the same length of RNA, probably through Ded1p oligomerization. Such ATP analog-induced association of additional Ded1p molecules likely takes place during duplex unwinding.

Eckhard Jankowsky (Advisor)
William Merrick (Committee Chair)
Alan Tartakoff (Committee Member)
Jeff Coller (Committee Member)
Michael Harris (Committee Member)
255 p.

Recommended Citations

Citations

  • Jia, H. (2011). Molecular Mechanism of the TRAMP Complex [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1310139879

    APA Style (7th edition)

  • Jia, Huijue. Molecular Mechanism of the TRAMP Complex. 2011. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1310139879.

    MLA Style (8th edition)

  • Jia, Huijue. "Molecular Mechanism of the TRAMP Complex." Doctoral dissertation, Case Western Reserve University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1310139879

    Chicago Manual of Style (17th edition)