Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo

摘要

The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S . pombe . 122 point mutation mutants were mapped to a recently solved 3.9 ? structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo , and surprisingly, amounts of transcription in vivo . Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. Author Summary RNA polymerase (RNAP) III is a multisubunit enzyme that synthesizes large amounts of transfer RNAs and a few other abundant short structural RNAs essential for the proliferation of all eukaryotic cells. To meet this demand, RNAP III must repeatedly initiate and terminate RNA synthesis with high efficiency, up to one million times on each tRNA gene per cell doubling. Much more research has been done on initiation than on termination. We performed a genetic screen focused on Rpc1, the largest RNAP III subunit, after subjecting it to random mutagenesis and identifying single amino acid substitutions that specifically affected its ability to terminate. Over 120 mutations were isolated and mapped onto a recently published, near-atomic resolution structure of RNAP III in synthesis mode. Mutations were concentrated in mobile elements of the active center of the enzyme, the bridge helix and the trigger loop, as well as regions that help recruit the RNA 3' cleavage domain of the C11 subunit which transiently interacts with the active center. The data provide evidence that RNAP III controls termination via its active center. Biochemical characterization demonstrated that a mutant RNAP III synthesizes RNA at a higher rate than wild type and also synthesizes more RNA in vivo .