Abstract
RNA polymerase (pol) III, assisted by the transcription factors TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete pol III transcription complexes (Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118-4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1- and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNA(Ile) ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c open reading frame, was found to be the strictly conserved remnant of an ancient tDNA(Arg). The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.
Highlights
We have shown that the S. cerevisiae ZOD1 locus encodes a novel pol III-transcribed, non coding RNA whose expression is subject to chromatin repression
Our data suggest that derepression of ZOD1 and ETC5 transcription upon nucleosome depletion involves activation of poised RNA polymerase III, rather than enhanced recruitment of transcription components, providing the first example of post-recruitment mechanism of pol III activation
We have traced the evolutionary history of ZOD1 back to a tDNAIle(AAT) ancestor that is still present and syntenic in K. lactis and A. gossypii
Summary
DNA Templates—The S. cerevisiae ZOD1 locus was amplified from yeast genomic DNA (strain S288C) using the high fidelity Pfu DNA polymerase (Promega) and the following oligonucleotide primers: ZOD1_fw, 5Ј-TTCGGGTGCCAATTTAGCGTGC-3Ј; ZOD1_rev, 5Ј-AGTGTCGGCCACCTGATAAAGG-3Ј. The disruption cassette was obtained by PCR amplification of the kanMX coding sequence from the ⌬gcn strain of the yeast gene knock-out collection (Open Biosystems, Huntsville, AL) using the following oligonucleotide primers: ZOD1DEL_fw, 5Ј-CGATCTAACTATTCTGTAAATTCTGTAGTGACTCTTTAGGGCGTACGCTGCAGGTCGAC-3Ј; ZOD1DEL_rev, 5Ј-AAATGGAAAGGGAAAAATTAAGGCTGCGAAAATTAATAATAATCGATGAATTCGAGCTCG-3Ј. In Vivo Transcription Analyses—Total RNA was purified as described [18] from yeast cells grown to A600 ϭ 0.8 –1 in the appropriate selective medium and temperature. For detection of ETC5/RNA170 transcript, a double-stranded DNA probe was produced by random priming labeling using an ETC5 fragment amplified from yeast genomic DNA with the oligonucleotides ETC5_fw, 5ЈTAGCGTTACGTTCGATACCTTCAC-3Ј, and ETC5_rev Each culture was halved, spun down, and resuspended: half in galactose-containing medium and half in glucose-containing medium These cultures were incubated for different time periods at 30 °C and subjected to micrococcal nuclease (MNase) treatment, as follows. 12.5 l of Platinum SYBR Green qPCR SuperMix UDG (Invitrogen)
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