Mutational Analysis of the Hydrolytic Activity of Yeast RNA Polymerase III

Ekaterina V Bobkova,Natasha Habib,Gemma Alexander,Benjamin D Hall

doi:10.1074/jbc.274.30.21342

Ekaterina V Bobkova, Natasha Habib + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.30.21342

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Abstract

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309-325 gave slightly elevated nuclease activity. In region 367-376, two mutations gave 12-15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056-9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli beta-subunit sequences between Rif(r) clusters I and II, produce changes in the relative yields of A- and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T(5) terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3'-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.

Highlights

The elongation phase of transcription by DNA-dependent RNA polymerases has long been viewed as a repetitive sequence of choices between two alternative outcomes: addition of another nucleotide to the growing RNA or dissociation of enzyme and RNA from the DNA template, resulting in termination [2]
These mutant polymerases read through the U5 tract in SUP4⌬94 that is placed at the location of the normal U7GU6 terminator, producing abnormally long pre-tRNAs that are not matured to functional tRNATyr
RNA polymerase III preparations isolated from several yeast strains bearing increased or decreased termination mutations have an altered response to several U-rich pause sites within the SUP4 template [11]

Summary

Introduction

The elongation phase of transcription by DNA-dependent RNA polymerases has long been viewed as a repetitive sequence of choices between two alternative outcomes: addition of another nucleotide to the growing RNA or dissociation of enzyme and RNA from the DNA template, resulting in termination [2]. Mutant polymerases with increased read-through of an intronic U5 sequence in the SUP4 UIV allele can efficiently produce biologically active suppressor tRNATyr [9, 10] These mutant polymerases read through the U5 tract in SUP4⌬94 that is placed at the location of the normal U7GU6 terminator, producing abnormally long pre-tRNAs that are not matured to functional tRNATyr. By in vivo mutant screening with these same two SUP4 alleles, a large number of second-largest subunit mutations having the opposite phenotype were obtained. Qualitative effects upon the release of pGpU relative to pUpU were observed for mutations in the region from amino acids 476 to 485 (analogous to Rifr cluster II of E. coli rpoB [13])

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