Analysis of Gene Induction and Arrest Site Transcription in Yeast with Mutations in the Transcription Elongation Machinery

Megan Wind-Rotolo,Daniel Reines

doi:10.1074/jbc.m011322200

Abstract

In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands. Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles. There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthrough in vivo. Saccharomyces cerevisiae strains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs. Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation. Both mutants and a combined double mutant were defective in induction of GAL1 and ENA1. This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants. These data are consistent with mutants in the Elongator complex, which show slow inductive responses. When a potent in vitro arrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation. These data suggest that an alternative elongation surveillance mechanism exists in vivo to overcome arrest.

Highlights

Several factors have been identified that act as transcription elongation factors in vitro
Induction of ENA1 Transcription Is Impaired in Elongation Mutants—Deletion of the Elongator subunit genes ELP1 and ELP3 result in delayed transcriptional induction responses
We provide evidence that mutations in the elongation machinery negatively impact the ability of yeast to carry out two transcriptional induction processes, consistent with prior reports describing the role of other elongation factors in gene induction and mRNA biosynthesis in yeast (5, 9 –12)

Summary

Gene Induction and Arrest Site Usage in Elongation Mutants

Of this system is that the biochemical consequence of this mutation has been characterized as has the mechanism by which SII relieves arrest in vitro [2]. The effect of 6AU on transcriptional responses, which is expected to slow elongation rates and promote arrest in vivo, was assessed in wild-type and mutant cells. We measured the efficiency of transcription through a strong, well-characterized in vitro arrest site for yeast pol II in the presence and absence of 6AU. That arrest sites defined in vitro were not effective in vivo may reflect the fact that this type of elongation impediment is not rate-limiting in vivo. SII and an efficiently elongating pol II have general effects in augmenting transcription in vivo

EXPERIMENTAL PROCEDURES

RESULTS

TABLE I Strains used

DISCUSSION