SUMMARY Pre-mRNA 3′-end formation is an essential step in eukaryotic gene expression. Over half of human genes produce alternatively polyadenylated mRNAs, suggesting that regulated polyadenylation is an important mechanism for post-transcriptional gene control. Although a number of mammalian mRNA 3′ processing factors have been identified, the full protein composition of the 3′ processing machinery has not been determined, and its structure is unknown. Here we report the purification and subsequent proteomic and structural characterization of human mRNA 3′ processing complexes. Remarkably, the purified 3′ processing complex contains ~85 proteins, including known and new core 3′ processing factors and over 50 proteins that may mediate crosstalk with other processes. Electron microscopic analyses show that the core 3′ processing complex has a distinct “kidney” shape and is ~250 Å in length. Together, our data has revealed the complexity and molecular architecture of the pre-mRNA 3′ processing complex.

Function Ca2ϩ pumps, together with Ca 2ϩ release channels, form ubiquitous Ca 2ϩ regulatory systems in muscle and non-muscle cells. The sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCA) 1 and the plasma membrane Ca 2ϩ -ATPases have the highest affinity for Ca 2ϩ removal from the cytoplasm and, together, set resting cytoplasmic Ca 2ϩ concentrations. Three differentially expressed genes encode SERCA proteins (1). SERCA1a and -1b are expressed in fast-twitch skeletal muscle, but loss of SERCA1 function in Brody disease is sufficiently compensated to preserve life (2). SERCA2a is the cardiac/slow-twitch isoform, whereas SERCA2b, with a C-terminal extension, is expressed in smooth muscle and non-muscle tissues. It is almost certainly an essential gene. SERCA3 is expressed in a limited set of non-muscle tissues, including endothelial, epithelial, and lymphocytic cells and platelets, and its knockout is not lethal (3).SERCA enzymes are typical of the class of P-type ATPases, which form a phosphoprotein intermediate and undergo conformational changes during the course of ATP hydrolysis (4, 5). Some of the conformational states can be stabilized, either by adjustment of reaction conditions or through mutagenesis, and characterized as intermediates in the overall reaction cycle (Fig. 1A). The phosphorylated intermediate, E 1 P(Ca) 2 , can phosphorylate ADP, whereas E 2 P can only react with water. The formation of E 1 P requires that two high affinity Ca 2ϩ binding sites be occupied. The enzyme is then phosphorylated by ATP and, concomitantly, the two Ca 2ϩ ions are occluded and can no longer exchange with cytoplasmic Ca 2ϩ . The rate-limiting transition to E 2 P is accompanied by loss of Ca 2ϩ into the lumen, the affinity having fallen by 3 orders of magnitude. Hydrolysis of E 2 P and regeneration of the high affinity Ca 2ϩ binding sites (E 1 (Ca) 2 ) complete the reversible cycle. High lumenal Ca 2ϩ drives the formation of E 1 P from phosphate (P i ), and its effect on the level of E 1 P led Jencks (5, 6) to postulate a second set of Ca 2ϩ

Summary The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14 MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and mRNP remodeling machinery right over the NPC's central channel, rather than on distal cytoplasmic filaments as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Bacterial GreA and GreB promote transcription elongation by stimulating an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. The structure of Escherichia coli core RNA polymerase bound to GreB was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 A, allowing fitting of high-resolution RNA polymerase and GreB structures. In the resulting model, the GreB N-terminal coiled-coil domain extends 45 A through a channel directly to the RNA polymerase active site. The model leads to detailed insights into the mechanism of Gre factor activity that explains a wide range of experimental observations and points to a key role for conserved acidic residues at the tip of the Gre factor coiled coil in modifying the RNA polymerase active site to catalyze the cleavage reaction. Mutational studies confirm that these positions are critical for Gre factor function.