Judith A. Jaehning, Ph.D.

Selected Abstracts

A multiplicity of mediators: alternative forms of transcription complexes communicate with transcriptional regulators.

Chang M, Jaehning JA.

Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80262, USA.

The already complex process of transcription by RNA polymerase II has become even more complicated in the last few years with the identification of auxiliary factors in addition to the essential general initiation factors. In many cases these factors, which have been termed mediators or co-activators, are only required for activated or repressed transcription. In some cases the effects are specific for certain activators and repressors. Recently some of these auxiliary factors have been found in large complexes with either TBP, as TBP-associated factors (TAFs) in the general factor TFIID, or with pol II and a subset of the general factors, referred to as the 'holoenzyme'. Although the exact composition of these huge assemblies is still a matter of some debate, it is becoming clear that the complexes themselves come in more than one form. In particular, at least four forms of TFIID have been described, including one that contains a tissue-specific TAF and another with a cell type-specific form of TBP. In addition, in yeast there are at least two forms of the 'holoenzyme' distinguished by their mediator composition and by the spectrum of transcripts whose expression they affect. Genetic and biochemical analyses have begun to identify the interactions between the components of these complexes and the ever increasing family of DNA binding regulatory factors. These studies are complicated by the fact that individual regulatory factors often appear to have redundant interactions with multiple mediators. The existence of these different forms of transcription complexes defines a new target for regulation of subsets of eukaryotic genes.

Nucleic Acids Res 1997 Dec 15;25(24):4861-5

A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling.

Chang M, French-Cornay D, Fan HY, Klein H, Denis CL, Jaehning JA.

Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160-1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Delta ccr4Delta, paf1Delta hpr1Delta, ccr4Delta hpr1Delta, and ccr4Delta gal11Delta double mutants. In addition, paf1Delta and ccr4Delta are lethal in combination with srb5Delta, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Delta, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Delta and ccr4Delta mutations, as well as gal11Delta, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Delta mpk1Delta and paf1Delta pkc1Delta double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Delta strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes.

Mol Cell Biol 1999 Feb;19(2):1056-67

Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.

Cliften PF, Jang SH, Jaehning JA.

Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver 80262, USA.

Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme.

Mol Cell Biol 2000 Sep;20(18):7013-23