MUTANT ANALYSIS OF TERMINATION AND ELONGATION BY RNA POLYMERASE III

Abdulsalam  A. SHAABAN

In this study, I have analyzed the roles in elongation and termination by yeast RNA polymerase III of four regions of the RET1 gene product, the second largest subunit of the enzyme. I isolated a large set of ret1 mutants altered in polymerase III termination by genetic screening of pools of random mutations created by oligonucleotide‑directed in vitro mutagenesis. The screen detects increases in ochre suppression of the  ade 2‑1₀  locus due to more efficient production of a correct length SUP4 RNA from partially defective SUP4  tRNA ^Tyr allele. All four regions yielded increased termination mutations, while only the first three gave reduced termination mutations.

I have characterized the in vitro elongation and termination properties of RNA polymerases isolated from a few phenotypically strong mutants. These studies have shown that mutations in region 300‑325 do not alter the elongation behavior of the polymerase at non‑terminator DNA sites. Mutations in regions 455‑486,487‑521, and 1061‑1082, however, alter the elongation properties of the enzyme. Increased efficiency of termination phenotype caused by mutations in these regions correlates with increased dwell times by the mutant enzyme at non‑terminator pause sites on the SUP4 DNA template. Some reduced termination mutants in regions 455-486 and 487‑521 have elongation properties similar to the wild‑type enzyme, while others have peculiar patterns of pausing; they show longer dwell times at a subset of the in vitro observed pause sites and normal dwell times at others.

The nature of the termination phenotypes (increased or reduced) produced by mutations in the regions studied, the strengths of the phenotypes, and the in vitro elongation properties of the corresponding mutant RNA polymerases led me to suggest that these regions are part of three separate functional domains. Regions 300‑325 likely facilitates termination by having a positive role in RNA release; both regions 455‑486 and 487‑521 are possibly involved in the recognition and response to the termination signal in the RNA and/or DNA; region 1061‑1082 is likely involved in a step of the transcription reaction that is important for optimal chain elongation