Functional Analysis of the Ninth Subunit of Yeast RNA Polymerase II, RPB9

Abstract

<p>RNA polymerase II is the eukaryotic enzyme that synthesizes mRNA. It is a complex enzyme that is highly regulated by many protein factors throughout transcription. RNA polymerase II comprises twelve subunits, each of which likely plays a specific role in the function of the enzyme. The ninth subunit, RPB9, is involved in the initiation and elongation stages of transcription. In yeast, deletion of RPB9 results in sensitivity to high and low temperatures, as well as to the drug 6-aza-uracil. RPB9-deficient strains exhibit an alteration in the selection of transcript start sites, with an upstream shift of the start site window. The RNA polymerase II isolated from RPB9-null strains is unable to recognize intrinsic pause sites during elongation. The polymerase molecules that do arrest cannot resume transcribing upon stimulation by the elongation factor TFIIS. RPB9 is a small polypeptide, containing two zinc-binding regions (Zn 1 and Zn2) connected by an intervening sequence (linker region). This study presents the results of a mutational analysis designed to assisn th various functions of RPB9 to regions within the subunit. The Zn1 region was shown to be required for the start site selection activity of RPB9. When expressed in an RPB9-null yeast strain, the Zn1 region was found to be sufficient to restore start site selections to the wildtype pattern. Using a gel shift assay and purified proteins, an RNA polymerase II binding region was located to a conserved sequence (D---DPTLPR) within the linker region. The elongation activity of RPB9 was ascribed to the Zn2 region. Conserved charged residues within Zn2 were essential for the ability of RPB9 to mediate the reactivation of RNA polymerase II upon stimulation by TFIIS. This region of Zn2 is analogous to a charged flexible loop within the zinc ribbon domain of TFIIS. These observations provide the basis for a preliminary model of RPB9 interactions with RNA polymerase II.</p>