The structure of the Gla‐domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)‐like domain modified by a large insert containing an additional disulfide, followed by a typical EGF‐like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin‐like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin‐like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non‐primed side.

The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.

General transcription factor Sll, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the Sll p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the Sll p1lO and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.Eukaryotic messenger RNA synthesis is a complex biochemical process controlled in part by the concerted action of a set of general transcription factors that regulate the activity of RNA polymerase II at both the initiation (1) and elongation (2, 3) stages of transcription. At least six general initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) have been identified in eukaryotic cells and found to promote selective binding of RNA polymerase II to promoters and to support a basal level of transcription (1). In addition, three general elongation factors (SII, TFIIF, and Sll) have been identified in eukaryotic cells and defined biochemically. The elongation factors fall into two functional classes.The sole member of the first class is SII. SII is an -38-kDa protein originally identified and purified to homogeneity by Natori and coworkers (4). SII binds to RNA polymerase II and promotes readthrough by polymerase through a variety of transcriptional impediments, including attenuation sites found in such genes as the human histone H3.3 (5, 6), adenovirus 2 major late (AdML) (7-9), and adenosine deaminase genes (10), as well as DNA-bound proteins (11) and drugs (12). SII is believed to promote readthrough by an unusual mechanism involving the reiterative shortening and reextending of nascent transcripts. Interaction of SIT with the RNA polymerase II elongation complex activates a latent ribonuclease activity that shortens nascent transcripts from their 3' ends (13,14 templates (16, 24). In this report, we describe the isolation, structure, and expression of a full-length cDNA encoding the functional SIII p18 subunit,** which is shown to be a ubiquitin homolog that belongs to a growing ubiquitin homology (UbH) gene family.In addition, we demonstrate that p18 functions as a dissociable regulatory subunit that is capable of activating overall SIII transcriptional activity through interactions with the SIII pl 10 and p15 subunits. MATERIALS AND METHODSIsolation of a cDNA Encoding the SIII p18 Subunit. SIII was purified to near homogeneity from rat liver nu...