Structure and function of the polymerase core of TRAMP, a RNA surveillance complex.
The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ss superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ss superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-A resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol beta RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3' end plays a role in substrate recognition.
Hamill
Stephanie
S
Departments of Cell Biology, and Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Wolin
Sandra L
SL
Reinisch
Karin M
KM
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; C415825:Air2 protein, S cerevisiae; D046912:Multiprotein Complexes; D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004259:DNA-Directed DNA Polymerase; C487545:PAP2 protein, S cerevisiae