Academic journal article Genetics

Mutations in the Saccharomyces Cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil

Academic journal article Genetics

Mutations in the Saccharomyces Cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil

Article excerpt

ABSTRACT

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the "lid" and "rudder," two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.

RNA polymerase II (RNAPII) is composed of 12 polypeptides with a high degree of structural conservation from yeast to humans (HAHN 2004). In Saccharomyces cerevisiae, Rpbl and Rpb2 form the central part of RNAPII and share an ample contact surface. The interaction between both subunits shapes several domains of the enzyme, such as the active center, which is formed by the "active site" and the "hybrid-binding" regions of Rpbl and Rpb2, respectively (CRAMER et al. 2001). The functional complexity of RNAPII is reflected in its structure, revealing the existence of a large number of regions with very specific functions. The core polymerase requires the association of a number of initiation factors for promoter recognition and initiation of RNA synthesis. Once in elongation mode, the crosstalk between RNAPII and a number of associated factors assures a proper mRNA synthesis (SiMS et al. 2004). To gain further insight into the process of transcription, we have isolated mutants of KPO21/KPB1, encoding the largest subunit of the RNAPII in yeast, obtained by a random mutagenesis. The mutants were selected by their sensitivity to 6-azauracil (6AU), a drug that decreases GTP and UTP pools (EXINGER and LACROUTE 1992). We reasoned that leaky mutants of RNAPII might be susceptible to imbalances in the intracellular nucleotide pools at steps in initiation, elongation, or termination.

Sensitivity to 6AU is a well-documented phenotype associated with transcription-elongation mutants. For example, the 6AUS mutant rpb2-10 (PlOl8S) is intrinsically arrest prone and has a slower polymerization rate (POWELL and REINES 1996; MASON and STRUHL 2005). Further, yeast knockouts of the genes DST1/PPR2 and SPT4, encoding the transcription elongation factors TFIIS (FiSH and KANE 2002) and Spt4 (HARTZOG et al. 1998), are highly sensitive to 6AU. As expected, the rpblE1230K (rpo21-24) point mutant, which decreases the binding of TFIIS to RNAPII (ARCHAMBAULT et al. 1992; Wu et al. 1996), is also 6AUS. Biochemical analyses have shown that TFIIS stimulates transcription elongation, increases the fidelity of incorporation of ribonucleotides, and is essential for the reactivation of arrested RNAPII in vitro (FiSH and KANE 2002). Apart from the well-characterized role of TFIIS in elongation in vitro, reports from different laboratories show that TFIIS is also involved in transcription initiation (DAVIE and KANE 2000; MALAGON et al. 2004; ADELMAN et al. 2005; PRATHER et al. 2005). Thus, it is possible that some of the sensitivity to 6AU in a dstl knockout is due to defects in initiation caused by lack of TFIIS, and it might be argued that some mutants in RNAPII that are sensitive to 6AU would be arrest prone or compromised in initiation of transcription. Although no single rpbl or rpb2 6AUS mutant is affected in initiation, the rpb2-101 (G369S) mutation is 6AUS and has an altered transcription initiation in the presence of a mutant TFIIB initiation factor (CHEN and HAMPSEY 2004). …

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