"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V max of the enzyme with little effect on the apparent K m for substrate, ATPMg 2؉ . Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K m for ATP-Mg 2؉ . Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels. Two types of adenylyl cyclase (AC)1 are ubiquitously expressed in mammalian cells, a well characterized gene family of transmembrane ACs (tmACs) and the recently discovered "soluble" AC (sAC). The tmACs are plasma membrane bound, and their activities are regulated by G proteins in response to extracellular stimuli such as neurotransmitters and hormones (reviewed in Ref. 1). In contrast, sAC is associated with various intracellular organelles, including mitochondria, centrioles, mitotic spindle, mid-bodies, and nuclei (2). sAC activity is modulated by bicarbonate (3) and, as shown in this report, by Ca 2ϩ ; regulation by these intracellular signaling molecules suggests that sAC mediates cAMP-dependent responses to intrinsic cellular changes (4, 5).The catalytic mechanism of tmACs has been determined from biochemical and crystallographic studies. tmACs convert ATP to cAMP using two-metal catalysis where one ion acts as a free metal and the other coordinates ATP in the active site (6, 7). Its activators, G␣ s subunit or forskolin, stimulate tmACs by allosteric modulation of the active site (8, 9). More than 25 years ago, when soluble AC activity was first discovered, it was predicted to be molecularly distinct from tmACs because its activity appeared to be dependent on the presence of the divalent cation, Mn 2ϩ , and it was insensitive to forskolin and G protein regulation (10 -12). These differential properties enabled purification (13) and cloning of sAC from rat testis (14). The sAC gene is indeed molecularly distinct from tmACs; it possesses no transmembrane domains, and its catalytic domains are more closely related to those of cyanobacterial ACs than to those from other eukaryotic ACs. The purified soluble AC exhibited ϳ10-fold lower affinity for substrate ATP relative to tmACs (tmAC K m for ATP-Mn 2ϩ is ϳ 100...

Chromatin is the physiological template for many nuclear processes in eukaryotes, including transcription by RNA polymerase II. In vivo, chromatin is assembled from genomic DNA, core histones, linker histones such as histone H1, and nonhistone chromatin-associated proteins. Histone H1 is thought to act as a general repressor of transcription by promoting the compaction of chromatin into higher-order structures. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the effects of histone H1 on estrogen receptor ␣ (ER␣)-mediated transcription with chromatin templates. We show that histone H1 acts as a potent repressor of ligand-and coactivator-regulated transcription by ER␣. Histone H1 exerts its repressive effect without inhibiting the sequence-specific binding of ER␣ to chromatin or the overall extent of targeted acetylation of nucleosomal histones by the coactivator p300. Instead, histone H1 acts by blocking a specific step in the ER␣-dependent transcription process, namely, transcription initiation, without affecting transcription reinitiation. Together, our data indicate that histone H1 acts selectively to reduce the overall level of productive transcription initiation by restricting promoter accessibility and preventing the ER␣-dependent formation of a stable transcription preinitiation complex.Chromatin is the physiological template for many nuclear processes in eukaryotes, including transcription, replication, and repair of the genome. The assembly of genomic DNA into chromatin has important functional consequences for the regulation of RNA polymerase II (pol II)-encoding genes since chromatin acts as a general repressor of transcription. Sequence-specific DNA-binding activator proteins, ATP-dependent chromatin-remodeling complexes, and coactivators are required to overcome chromatin-mediated transcriptional repression (23). Transcriptional activator proteins target chromatin-remodeling complexes and coactivators to specific promoters. Chromatin-remodeling complexes use the energy from ATP to reorganize chromatin at the promoters, which allows access of the promoter DNA to the basal transcriptional machinery (reviewed in reference 26). Coactivators have at least two roles (reviewed in references 35 and 53). First, those coactivators with intrinsic histone acetyltransferase (HAT) activity acetylate nucleosomal histones at the promoter, which is thought to facilitate chromatin remodeling. Second, coactivators can make contacts with the basal transcriptional machinery and RNA pol II to facilitate the formation of transcription preinitiation complexes (PICs). The concerted actions of the activators, chromatin-remodeling complexes, and coactivators lead to the transcription of genes assembled into chromatin (reviewed in references 16, 23, 26, 34, and 35).Chromatin is assembled in vivo from genomic DNA, core histones, linker histones, and nonhistone chromatin-associated proteins. The core histones form the structural core of the nucleosome (38), wherea...