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Direct interaction between the activation domain of the 289R E1A protein of adenovirus and the TATA box binding protein is crucial to the E1A activation mechanism. Conservative substitution effects at all of the 49 E1A activation domain residues were in the zinc finger region.

In eukaryotes, RNA polymerase II-driven transcription and processing results in the formation of numerous mRNA 3' isoforms that for any given gene may differ from one another by as little as a single nucleotide. These 3' isoforms can vary in physical properties that may affect their function and stability. Here, we outline a systematic framework to measure individual mRNA 3' isoform half-lives on a genome-wide level in S. cerevisiae. Our approach utilizes the Anchor-Away system to sequester RNA polymerase II (Pol II) in the cytoplasm followed by direct single-molecule RNA sequencing to generate a highly detailed view of 3' isoform stability under most physiological conditions without many of the adverse effects associated with commonly used alternative approaches.

In Saccharomyces cerevisiae , previous measurements of mRNA stabilities have been determined on a per-gene basis. We and others have recently shown that yeast genes give rise to a highly heterogeneous population of mRNAs thanks to extensive alternative 3' end formation. Typical genes can have fifty or more distinct mRNA isoforms with 3' endpoints differing by as little as one and as many as hundreds of nucleotides. In our recent paper [Geisberg et al. Cell (2014) 156: 812-824] we measured half-lives of individual mRNA isoforms in Saccharomyces cerevisiae by using the anchor away method for the rapid removal of Rpb1, the largest subunit of RNA Polymerase II, from the nucleus, followed by direct RNA sequencing of the cellular mRNA population over time. Combining these two methods allowed us to determine half-lives for more than 20,000 individual mRNA isoforms originating from nearly 5000 yeast genes. We discovered that different 3' mRNA isoforms arising from the same gene can have widely different stabilities, and that such half-life variability across mRNA isoforms from a single gene is highly prevalent in yeast cells. Determining half-lives for many different mRNA isoforms from the same genes allowed us to identify hundreds of RNA sequence elements involved in the stabilization and destabilization of individual isoforms. In many cases, the poly(A) tail is likely to participate in the formation of stability-enhancing secondary structures at mRNA 3' ends. Our results point to an important role for mRNA structure at 3' termini in governing transcript stability, likely by reducing the interaction of the mRNA with the degradation apparatus.

Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene-replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site-specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR-based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR- and oligonucleotide-based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene., (Copyright © 2013 John Wiley & Sons, Inc.)

In yeast, TFIID strongly associates with nearly all ribosomal protein (RP) promoters, but a TAF-independent form of TBP preferentially associates with other active promoters. RP promoters are regulated in response to growth stimuli, in most cases by a Rap1-containing activator. This Rap1-dependent activator is necessary and sufficient for TFIID recruitment, whereas other activators do not efficiently recruit TFIID. TAFs are recruited to RP promoters even when TBP and other general transcription factors are not associated, suggesting that TFIID recruitment involves a direct activator-TAF interaction. Most RP promoters lack canonical TATA elements, and they are preferentially activated by the Rap1-containing activator. These results demonstrate activator-specific recruitment of TFIID in vivo, and they suggest that TFIID recruitment is important for coordinate expression of RP genes.