A eukaryotic chromosomal origin of replication was identified in the yeast Saccharomyces cerevisiae. By several criteria, including map position, deletion analysis, and a synthetic form of saturation mutagenesis, the origin co-localized with the HMR-E silencer, which is a DNA element that represses transcription of the adjacent genes. A specific site within the silencer was required for both initiation of chromosomal replication and for repression of transcription. This analysis directly demonstrates that initiation of eukaryotic chromosomal replication is dependent on specific sequence elements and that a particular element can act in both initiation of chromosomal replication and regulation of transcription.
We have resolved a previously unidentified factor (TFIIID) that is required for in vitro transcription of polymerase III templates. Our ability to resolve factor D from each of the other components of the transcription machinery (polymerase and transcription factors IIIB and IIIC) allowed us to test the capacity of these separated components to form stable complexes with tRNA genes. We find that none of the individual components binds detectably to tRNA genes, but that certain combinations of transcription factors do bind. Our results show that TFIIID is essential for binding and that formation of a full transcription complex can proceed by either of two different pathways.
Heterochromatin resides near yeast telomeres and at the cryptic mating-type loci, HML and HMR, where it silences transcription of the a-and a-mating-type genes, respectively. Ku is a conserved DNA end-binding protein that binds telomeres and regulates silencing in yeast. The role of Ku in silencing is thought to be limited to telomeric silencing. Here, we tested whether Ku contributes to silencing at HML or HMR . Mutant analysis revealed that yKu70 and Sir1 act collectively to silence the mating-type genes at HML and HMR. In addition, loss of yKu70 function leads to expression of different reporter genes inserted at HMR. Quantitative chromatin-immunoprecipitation experiments revealed that yKu70 binds to HML and HMR and that binding of Ku to these internal loci is dependent on Sir4. The interaction between yKu70 and Sir4 was characterized further and found to be dependent on Sir2 but not on Sir1, Sir3, or yKu80. These observations reveal that, in addition to its ability to bind telomeric DNA ends and aid in the silencing of genes at telomeres, Ku binds to internal silent loci via protein-protein interactions and contributes to the efficient silencing of these loci.
194 -199). In contrast, the sequence elements that may comprise origins in multicellular eukaryotes are largely unknown. The yeast HMR-E region is both a chromosomal origin of replication and a silencer that represses transcription of adjacent genes through a position effect. The analysis presented here indicated that HMR-E had a novel DNA structure that was more complex than defined for other yeast origins, and thus revealed that there is variation in the structural complexity of yeast origins. In contrast to "simple" yeast origins, the origin at HMR-E consisted of at least three independent subregions that had the capacity to initiate replication. We have termed HMR-E a compound origin to reflect its structural complexity. Furthermore, only one origin within the compound origin was a silencer.
Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMR a-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMR a-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the·temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.