New structures of RNA polymerase II (pol II) transcribing complexes reveal a likely key to transcription. The trigger loop swings beneath a correct nucleoside triphosphate (NTP) in the nucleotide addition site, closing off the active center and forming an extensive network of interactions with the NTP base, sugar, phosphates, and additional pol II residues. A histidine side chain in the trigger loop, precisely positioned by these interactions, may literally "trigger" phosphodiester bond formation. Recognition and catalysis are thus coupled, ensuring the fidelity of transcription.

Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to alpha-amanitin, indicating a functional interaction between His1085 and alpha-amanitin that is supported by rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct alpha-amanitin interference with the TL.

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo , Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo , implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process.

The cyclooxygenase (COX)-2 enzyme is responsible for increased prostaglandin formation in inflammatory states and is the major target of nonsteroidal anti-inflammatory drugs. Normally COX-2 expression is tightly regulated, however, constitutive overexpression plays a key role in colon carcinogenesis. To understand the mechanisms controlling COX-2 expression, we examined the ability of the 3-untranslated region of the COX-2 mRNA to regulate post-transcriptional events. When fused to a reporter gene, the 3-untranslated region mediated rapid mRNA decay (t1 ⁄2 ‫؍‬ 30 min), which was comparable to endogenous COX-2 mRNA turnover in serum-induced fibroblasts treated with actinomycin D or dexamethasone. Deletion analysis demonstrated that a conserved 116-nucleotide AU-rich sequence element (ARE) mediated mRNA degradation. In transiently transfected cells, this region inhibited protein synthesis approximately 3-fold. However, this inhibition did not occur through changes in mRNA stability since mRNA half-life and steady-state mRNA levels were unchanged. RNA mobility shift assays demonstrated a complex of cytoplasmic proteins that bound specifically to the ARE, and UV cross-linking studies identified proteins ranging from 90 to 35 kDa. Fractionation of the cytosol showed differential association of ARE-binding proteins to polysomes and S130 fractions. We propose that these factors influence expression at a post-transcriptional step and, if dysregulated, may increase COX-2 protein as detected in colon cancer.Arachidonic acid metabolites, particularly prostaglandins, participate in both normal growth responses and in aberrant growth, including carcinogenesis (1-3). The committed step in the conversion of free arachidonic acid to prostaglandins is catalyzed by cyclooxygenase (COX), 1 also termed prostaglandin H synthase (4). There are two isoforms of cyclooxygenase; the type 1 (COX-1) isoform is present under resting conditions in many cells, and presumably makes prostaglandins for physiological functions. The type 2 (COX-2) is not normally present under basal conditions, or present in very low amounts, but is rapidly induced by cytokines, growth factors, and tumor promoters to result in prostaglandin synthesis associated with inflammation and carcinogenesis (5-8).Insight into the molecular events controlling COX-2 expression preceded its discovery. Early studies of the regulation of inducible COX activity identified time-dependent modulation of transcriptional and post-transcriptional phases of the COX biosynthetic pathway (9). Since the molecular cloning and characterization of COX-2, extensive studies identified transcriptional regulation of COX-2 (10 -13). COX-2 also may be regulated at the post-transcriptional level since the 3Ј-untranslated region (3Ј-UTR) of its mRNA contains multiple copies of adelylate-and uridylate-rich (AU-rich) elements (AREs) composed of the sequence 5Ј-AUUUA-3Ј. This element, which is present within the 3Ј-UTRs of many proto-oncogene and cytokine mRNAs, confers post-transcriptional control of e...