Partial Truncation of the Yeast RNA Polymerase II Carboxyl-terminal Domain Preferentially Reduces Expression of Glycolytic Genes

Eytan Meisels,Opher Gileadi,Jeffry L Corden

doi:10.1074/jbc.270.52.31255

Abstract

The largest subunit of RNA polymerase II contains an essential carboxyl-terminal domain (CTD) that consists of highly conserved heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Yeast cells with a partially truncated CTD grow slowly, are temperature- and cold-sensitive, and are unable to fully activate transcription of some genes. Screening a yeast wild-type cDNA library by means of comparative hybridization we find that CTD truncation preferentially reduces transcription of genes encoding glycolytic enzymes. Using a newly developed dual reporter assay we demonstrate that sensitivity to CTD truncation is conferred by the glycolytic gene promoters. Expression driven by glycolytic gene promoters is reduced, on average, about 3-fold in strains with the shortest CTD growing on either fermentable or nonfermentable carbon sources. Sensitivity to CTD truncation is particularly acute for the constitutively expressed ENO1 gene, which is reduced 10-fold in a strain with only eight CTD repeats. The sensitivity of constitutive ENO1 expression argues that CTD truncation can cause defects in uninduced as well as induced transcription.

Highlights

The carboxyl-terminal domain (CTD)1 of the RNA polymerase II largest subunit consists of tandem repeats of nearly identical amino acid heptamers [1, 2]
Identification of Genes Sensitive to CTD Truncation— previous studies have shown that INO1 and GAL10 are not fully induced in short CTD strains (8 –10), this defect cannot explain the slow growth of these strains in rich medium
Yeast CTD truncation mutants growing in rich medium have progressively more severe growth defects as the length of the CTD approaches the minimum length required for viability [3, 7, 11]

Summary

Introduction

The carboxyl-terminal domain (CTD) of the RNA polymerase II largest subunit consists of tandem repeats of nearly identical amino acid heptamers [1, 2]. Activated transcription driven by UAS elements in the INO1 and GAL10 promoters is defective in yeast CTD truncation strains (8 –10). In the shortest CTD strain tested (11 repeats) INO1-driven expression is reduced more than 10-fold, whereas GAL10-driven expression is reduced 2-fold [9] Uninduced transcription of these two genes was found to be unaffected by CTD truncation. CTD truncation in yeast produces a cold-sensitive lethal phenotype that can be suppressed by mutations in SRB genes [11]. Nine of these genes have been identified, and their products have been shown to form a complex with RNA polymerase II [14]. We propose that reduced transcription of highly expressed genes like ENO1 and ADH1 could lead to the growth defects associated with CTD truncation

Methods

Results

Conclusion