Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Gralla, J.D. (1980) Biochemistry 19, 3245-3253]. Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces. In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and is less likely following incorporation of GMP into the RNA message. Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded throughout the entire message region. This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling. Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling. Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Two proteolytically modified forms of T7 RNA polymerase have been characterized with respect to transcription initiation and processivity. One species, denoted 80K-20K, is singly cleaved within the region of the polypeptide chain between amino acids 172 and 180. The second species, denoted 80K, is generated by extensive proteolysis of the N-terminal 20K domain by trypsin. The 80K-20K form is fully active in initiation and escape from abortive cycling. It is deficient only in processivity on long DNA templates. Likewise, the 80K species shows initiation kinetics and abortive product synthesis similar to those of the native enzyme. This latter species, however, is unable to escape abortive cycling and shows no synthesis of transcripts longer than about eight bases. Studies of RNA and DNA binding to the three different forms of the enzyme by gel retention assays reveal that the native (98K), the 80K-20K, and the 80K species all form specific complexes with promoter-containing DNA. In addition, the native enzyme binds nonspecifically to double-stranded DNA, while the 80K-20K and 80K enzymes do not. The native enzyme also binds RNA. This RNA binding is reduced in the 80K-20K enzyme and is absent in the 80K species. We suggest a model for T7 RNA polymerase wherein the 20K N-terminal domain of the protein or a shared region between the N- and the C-terminal domains of the protein forms a nonspecific polynucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA [Basu, S., & Maitra, U. (1986) J. Mol. Biol. 190, 425-437. Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. Both of these reagents have drawbacks; DNase I is a bulky reagent and so provides low resolution, and (methidiumpropyl-EDTA)-Fe(II) intercalates into DNA and is therefore biased toward cleavage of double-stranded DNA. In this study, the interaction between the polymerase and the promoter has been probed with Fe(II)-EDTA. This reagent generates reactive hydroxyl radicals free in solution, which produces a more detailed picture of the polymerase-promoter complex. Two protected regions are observed on each of the two promoter DNA strands: from position -17 to position -13 and from position -7 to position -1 on the coding strand and from position -14 to position -9 and from position -3 to position +2 on the noncoding strand. From this pattern it is clear that if recognition occurs via double-stranded B-form DNA, then the protected regions lie on one face of the DNA helix, and therefore the enzyme must interact predominantly from one side of the DNA helix. Digestion of the DNA in a polymerase-promoter complex with a single-strand-specific endonuclease shows that a small region of the noncoding strand near position -5 is susceptible to cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin (1-170) ) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C 18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin (1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin (1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (K i ‫؍‬ 0.1 nM), human tissue kallikrein (K i ‫؍‬ 0.1 nM), human plasma kallikrein (K i ‫؍‬ 0.3 nM) and human factor XIa (K i ‫؍‬ 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (K i ‫؍‬ 1.6 M), factor IXa (K i ‫؍‬ 206 nM), factor Xa (K i ‫؍‬ 364 nM), and factor XIIa (K i ‫؍‬ 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin (1-170) prolonged the clotting time in an activated partial thromboplastin time assay.Blood clotting, resulting either from the extrinsic pathway following tissue injury or the intrinsic pathway following contact activation, involves tightly regulated proteolytic cascades (1). The intrinsic pathway is initiated by activation of factor XII either through proteolysis or contact with negatively charged surfaces. Activated factor XIIa, in turn, converts plasma prekallikrein to kallikrein, which can then activate additional factor XII. Factor XIIa activates factor XI, which, in turn, activates factor IX. Activated factor IX forms a complex with factor VIIIa, phospholipid, and calcium, which converts factor X to factor Xa. Factor X is also activated by the factor VIIatissue factor complex operating within the extrinsic pathway. Thrombin generation by factor Xa in complex with factor Va leads ultimately to the formation of the fibrin clot. Thrombus formation is also regulated by the fibrinolytic system whereby plasmin, formed from plasminogen by the action of kallikrein, tissue plasminogen activator (tPA), 1 or urokinase, breaks down both fibrinogen and fibrin (2).Protease inhibitors play critical roles in the regulation of the coagulation and fibrinolytic systems. Tissue factor pathway i...

Interrogation of the public expressed sequence tag (EST) data base with the sequence of preproaprotinin identified ESTs encoding two potential new members of the Kunitz family of serine protease inhibitors. Through reiterative interrogation, an EST contig was obtained, the consensus sequence from which encoded both of the novel Kunitz domains in a single open reading frame. This consensus sequence was used to direct the isolation of a full-length cDNA clone from a placental library. The resulting cDNA sequence predicted a 252-residue protein containing a putative NH 2 -terminal signal peptide followed sequentially by each of the two Kunitz domains within a 170-residue ectodomain, a putative transmembrane domain, and a 31-residue hydrophilic COOH terminus. The gene for this putative novel protein was mapped by use of a radiation hybrid panel to chromosome 19q13, and Northern analysis showed that the corresponding mRNA was expressed at high levels in human placenta and pancreas and at lower levels in brain, lung, and kidney. An endogenous soluble form of this protein, which was designated as placental bikunin, was highly purified from human placenta by sequential kallikrein-Sepharose affinity, gel filtration, and C 18 reverse-phase chromatography. The natural protein exhibited the same NH 2 terminus as predicted from the cloned cDNA and inhibited trypsin, plasma kallikrein, and plasmin with IC 50 values in the nanomolar range.The Kunitz (1, 2), Kazal (2), Serpin (3), and mucus (4) families of biological serine protease inhibitors play a vital role in the spatial and temporal regulation of in vivo proteolysis. The prototypical Kunitz inhibitor, bovine aprotinin (2), is a 58-amino acid protein containing three intrachain disulfide bonds in a spacing that is conserved in all family members (1, 2). Although the physiologic function of aprotinin is uncertain, it is a potent inhibitor of several serine proteases, and its potency against kallikrein and plasmin (5) may be relevant to its clinical mode of action (5, 6), particularly in the reduction of perioperative blood loss. A human functional homolog of aprotinin has not been identified, although several larger human proteins containing one or more Kunitz domains are known. These include: tissue factor pathway inhibitor (TFPI), 1 which contains three Kunitz domains (7) and inhibits both factor Xa and the factor VIIa-tissue factor complex (8); TFPI-2 (9), which contains two Kunitz domains (a bikunin) and is a potent inhibitor of the factor VIIa-tissue factor complex, factor XIa, and plasmin (10); and inter-␣-trypsin inhibitor, a plasma-associated bikunin (11). In addition, the following proteins are known to contain a single Kunitz domain: COL␣3/VI, the ␣(3) chain of type VI collagen (12); HKI-B9, a human Kunitz inhibitor (13); the membrane-associated amyloid precursor proteins APP 751 (14) and APP 770 (15); the amyloid precursor-like proteins (APLP) such as APLP2 (16). To identify novel human homologs of aprotinin we employed a bioinformatic approach that exploited the...