Identifier

etd-10302015-120951

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

RNA polymerase III (Pol III) transcribes tRNAs as well as other small non-coding RNAs. tRNA genes contain internal promoter sequences (A- and B-boxes) which can be specifically recognized and bound by TFIIIC. Binding of TFIIIC facilitates TFIIIB recruitment, which in turn targets Pol III to be recruited and initiate transcription. In addition to typical tRNA genes, there are other chromosomal regions, referred as extra-TFIIIC (ETC) sites, that are only bound by TFIIIC. Apart from transcription of genes, both complete and partially assembled Pol III complexes perform extra-transcriptional functions such as influencing nearby Pol II transcription, displacement of nearby nucleosomes, as well as chromatin boundary activities. By analyzing transcriptome data from high throughput RNA sequencing, we observed numerous alterations in intergenic transcription in close proximity to tDNAs and other Pol III complex binding sites after TFIIIC binding was globally compromised. Reduction of TFIIIC binding activity was achieved by using a yeast strain containing a mutation in the Reb1p binding site within the TFC6 promoter, which drastically reduces the level of the TFIIIC component Tfc6p. Analysis of loci adjacent to Pol III complex binding sites reveal both 5’- and 3’-extended transcripts, readthrough transcripts, and increased intergenic cryptic transcription. Many of the effects of 5’-UTR extension and de-repression appear to be due to the release of bidirectional activity of neighboring promoters. Translation of affected mRNAs is greatly altered because of the usage of upstream transcriptional start site (TSSs) at both TFC6 and TRM12 loci. The results presented here add another type of boundary activity to the known list of extra-transcriptional functions – the blocking activity of transcription from bidirectional promoters. Also, such activities might explain a function of the conserved ETC sites in yeast Saccharomyces cerevisiae. Analysis of the TFC6 locus suggests regulatory effects of assembled Pol III complexes at ETC6 site. Also, Reb1p is confirmed to be the transcriptional factor that binds and activates the TFC6 promoter and it binds several base pairs upstream of the ETC6 site. Analysis of these two divergently transcribed genes (ESC2 and TFC6) reveals that both have 5’-UTR extensions after Reb1p is depleted or the Reb1 consensus binding site is mutated, which is consistent with results from our transcriptome data. Similarly, altered TSS results in a significant decrease of the translational level of both TFC6 and ESC2. By assessing nucleosome content within the intergenic region ESC2-TFC6, we find that nucleosome positioning is slightly altered, which could explain the observed 5’-extended transcripts of both genes. Taken together, both of these studies demonstrate significant effects of assembled Pol III complexes and Reb1p binding on the transcription of neighboring Pol II promoters and on the translation of their mRNA products.

Date

2015

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Donze, David

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