Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II.
The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAP(II)) form a heterodimer that protrudes from the 10-subunit core of the enzyme. We have obtained crystals of the human Rpb4/Rpb7 heterodimer and determined the structure to 2.7 A resolution. The presence of putative RNA-binding domains on the Rpb7 subunit and the position of the heterodimer close to the RNA exit groove in the 12 subunit yeast polymerase complex strongly suggests a role for the heterodimer in binding and stabilizing the nascent RNA transcript. We have complemented the structural analysis with biochemical studies directed at dissecting the RNA-binding properties of the human Rpb4/Rpb7 complex and that of the homologous E/F complex from Methanocaldococcus jannaschii. A number of conserved, solvent-exposed residues in both the human Rpb7 subunit and the archaeal E subunit have been modified by site-directed mutagenesis and the mutants tested for RNA binding by performing electrophoretic mobility shift assays. These studies have identified an elongated surface region on the corresponding face of both subunit E and Rpb7 that is involved in RNA binding. The area spans the nucleic acid binding face of the OB fold, including the B4-B5 loop, but also extends towards the N-terminal domain.
Meka
Hedije
H
Department of Biological Sciences, Imperial College London, SW7 2BW, UK.
Werner
Finn
F
Cordell
Suzanne C
SC
Onesti
Silvia
S
Brick
Peter
P
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment
D019843:Archaeal Proteins; D021122:Protein Subunits; D012313:RNA; C505957:POLR2D protein, human; C477608:POLR2G protein, human; D012319:RNA Polymerase II