Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse.

The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions.

Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin.

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.

A conserved clathrin assembly motif essential for synaptic vesicle endocytosis.

Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.

A role for the clathrin assembly domain of AP180 in synaptic vesicle endocytosis.

We have used the squid giant synapse to determine whether clathrin assembly by AP180 is important for synaptic vesicle endocytosis. The squid homolog of AP180 encodes a 751 amino acid protein with 40% sequence identity to mouse AP180. Alignment of squid AP180 with other AP180 homologs shows that amino acid identity was highest in the N-terminal inositide-binding domain of the protein and weakest in the C-terminal clathrin assembly domain. Recombinant squid AP180 was able to assemble clathrin in vitro, suggesting a conserved three-dimensional structure that mediates clathrin assembly despite the divergent primary sequence of the C-terminal domain. Microinjection of the C-terminal domains of either mouse or squid AP180 into the giant presynaptic terminal of squid enhanced synaptic transmission. Conversely, a peptide from the C-terminal domain of squid AP180 that inhibited clathrin assembly in vitro completely blocked synaptic transmission when it was injected into the giant presynaptic terminal. This inhibitory effect occurred over a time scale of minutes when the synapse was stimulated at low (0.03 Hz), physiological rates. Electron microscopic analysis revealed several structural changes consistent with the inhibition of synaptic vesicle endocytosis; peptide-injected terminals had far fewer synaptic vesicles, were depleted of coated vesicles, and had a larger plasma membrane perimeter than terminals injected with control solutions. In addition, the remaining synaptic vesicles were significantly larger in diameter. We conclude that the clathrin assembly domain of AP180 is important for synaptic vesicle recycling at physiological rates of activity and that assembly of clathrin by AP180 is necessary for maintaining a pool of releasable synaptic vesicles.

AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin.

Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.

Probing the phosphoinositide binding site of the clathrin assembly protein AP-2 with photoaffinity labels.

The relative binding specificities of the subunitsof bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tethered p-benzoyldihydrocinnamoyl-InsPn (BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPn analogs that sampled the interface between the water and lipid phases, and (iii) sn-1-O-acyl-linked PtdInsPn analogs that interacted with proteins penetrating the bilayer. The InsPn and PtdInsPn probes bind with highest selectivity and affinity to the two alpha subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa mu subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPn binding site of alpha-AP-2 prefers more highly phosphorylated species, and (iii) the protein-acyl chain interactions showed high capacity but low selectivity.

Regulation of AP-3 function by inositides. Identification of phosphatidylinositol 3,4,5-trisphosphate as a potent ligand.

As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC8PtdIns(3,4, 5)P3) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC8-PtdIns(3,4,5)P3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC8-PtdIns(3,4, 5)P3 that establish specificity. Dioctanoyl-phosphatidylinositol 3, 4-bisphosphate, which does not have a 5-phosphate, and 4, 5-O-bisphosphoryl-D-myo-inosityl 1-O-(1, 2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC8-PtdIns(3,4,5)P3 as inhibitors of clathrin assembly. Deacylation of DiC8-PtdIns(3,4,5)P3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4, 5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.

Clathrin binding and assembly activities of expressed domains of the synapse-specific clathrin assembly protein AP-3.

We separately expressed the 58-kDa C-terminal, 42-kDa middle, 16-kDa C-terminal, and 33-kDa N-terminal regions of AP-3 (also called F1-20, AP180, NP185, and pp155), and determined their clathrin binding and assembly properties. The 58-kDa C-terminal region of AP-3 is able to bind to clathrin triskelia and assemble them into a homogeneous population of clathrin cages and will also bind to preassembled clathrin cages. The 42-kDa central region of AP-3 can bind to both clathrin triskelia and to clathrin cages, but cannot assemble clathrin triskelia into clathrin cages. The 16-kDa C-terminal region of AP-3 can bind to clathrin cages, but cannot bind to clathrin triskelia or assemble clathrin triskelia into clathrin cages. The clathrin binding activities of the 42-kDa central region and 16-kDa C-terminal region are weaker than the corresponding activity of either the 58-kDa C-terminal region or full-length AP-3. Previous efforts had mapped a clathrin binding site within the N-terminal 33 kDa of AP-3 (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Morris, S. A., Schroder, S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) EMBO J. 12, 667-675). However, although the N-terminal 33 kDa of AP-3 is able to bind to clathrin triskelia (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26), it does not promote their assembly into clathrin cages (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. CHem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26) or bind to preassembled clathrin cages (Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26). It appears that the smallest functional unit that carries out all of the reported clathrin binding and assembly properties of AP-3, essentially as well as the full-length protein, is the 58-kDa C-terminal region.

Bacterially expressed F1-20/AP-3 assembles clathrin into cages with a narrow size distribution: implications for the regulation of quantal size during neurotransmission.

F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bacterially expressed F1-20/AP-3 to bind and assemble clathrin cages. We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble clathrin as efficiently as preparations of F1-20/AP-3 from bovine brain. This establishes that the clathrin assembly activity found in F1-20/AP-3 preparations from brain extracts is indeed encoded by the cloned gene for F1-20/AP-3. It also demonstrates that post-translation modification is not required for activation of the clathrin binding or assembly function of F1-20/AP-3. Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1-20/AP-3 reveals a strikingly narrow size distribution. This may be important for the regulation of quantal size during neurotransmission. We also express the 33 kD NH2-terminus of F1-20/AP-3 in E. coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages. It has been suggested that the 33 kD NH2-terminus of F1-20/AP-3 constitutes a clathrin binding domain. We find that the bacterially expressed 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly. The finding that the 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies. The finding that the 33 kD NH2-terminus of F1-20/AP-3 fails to bind to clathrin cages is novel and potentially important. It is clear from these experiments that the 33 kD NH2-terminus of F1-20/AP-3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated.

Inhibition of clathrin assembly by high affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP-3.

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 microM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 microM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 microM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 microM. Moreover, 10 microM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.

Characterization of a set of T7 RNA polymerase active site mutants.

We have evaluated the elongation rates, processivities, and abortive transcription characteristics of a set of T7 RNA polymerase mutants that map to the polymerase active site. The effects of these mutations on transcription are complex: they cause decreases in activity and processivity during both the processive and abortive phases of transcription and exhibit disproportionate decreases in activity and processivity on poly(dA).poly(dT) or poly(dT) versus poly(dG).poly(dC) templates. They also exhibit an increase in the proportion of slippage dependent poly(G) transcript synthesis during the initial stages of transcription. It is shown that these multiple, distinct effects on transcription can be attributed to decreases in the mutant enzymes' phosphodiester bond formation rates. Estimates of the decreases in these rates are derived from the measured transcript elongation rates and processivities of the mutant enzymes.

The thumb subdomain of T7 RNA polymerase functions to stabilize the ternary complex during processive transcription.

To examine the function of the thumb subdomain in bacteriophage T7 RNA polymerase we constructed a set of deletion mutants within this subdomain. These mutants exhibited reduced processivity during the processive, but not the abortive, stage of transcription. Reduced processivity was found to be due primarily to an increase in the processive ternary complex dissociation rate (destabilization of the processive ternary complex). The destabilization of the ternary complex does not appear to be due to a decrease in the affinity of the polymerase for the nascent RNA. These observations support the proposal that the thumb subdomain functions to stabilize the processive ternary complex during the processive (but not the abortive) stage of transcription, probably by wrapping around the template to prevent polymerase dissociation.

The synapse-specific phosphoprotein F1-20 is identical to the clathrin assembly protein AP-3.

F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE). F1-20 was cloned and characterized because of its synapse specificity. AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein. Here we present evidence that establishes the identity of F1-20 and AP-3. Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE. These monoclonal antibodies also specifically recognize the cloned F1-20 protein expressed in Escherichia coli. The anti-F1-20 monoclonal antibody (mAb) stains a bovine protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3. The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain clathrin-coated vesicle extract. AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs. Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin. Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine. This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged clathrin-binding domain and an acidic, anomalously migrating domain.

Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure.

In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.

Characterization of a novel synapse-specific protein. I. Developmental expression and cellular localization of the F1-20 protein and mRNA.

A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse-specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1-20. Immunohistochemical and biochemical analyses revealed that the F1-20 protein is synapse associated. Western blot analyses revealed that the F1-20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1-20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1-20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1-20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1-20 protein by immunohistochemistry, and of the F1-20 mRNA by in situ hybridization. We found that the F1-20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1-20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1-20 protein are found in the cortically organized regions of the brain. The highest levels of F1-20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1-20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1-20 mRNA between different nuclear groups.

Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein.

The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.

Isolation and characterization of mutant bacteriophage T7 RNA polymerases.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.

Model for the mechanism of bacteriophage T7 RNAP transcription initiation and termination.

Characterization of a mutant T7 RNA polymerase (RNAP) that is active on non-promoter templates but has lost the ability to selectively utilize the T7 promoter led to the finding that wild-type T7 RNAP initiates transcription at a high rate on non-promoter templates but that most (approximately 90%) of these initiation events lead to synthesis of dinucleotides only. The anomalously high activity of T7 RNAP on poly(dC) templates (relative to other non-promoter templates) is due to a reduction in the rate of transcription abortion after dinucleotide synthesis rather than an increase in initiation. Evidence is presented that the transition from abortive to processive transcription is associated with a conformational change in T7 RNAP. The stability of the nascent chain in a ternary complex is shown to increase with increasing chain length in the 2 to 14 base range even when the size of the complementary RNA-DNA hybrid remains constant and small (2 to 3 base-pairs). Two mutant polymerases that show increased release of transcripts during abortive transcription and a proteolytically nicked polymerase that exhibits reduced RNA binding are shown to have reduced ability to read-through a T7 RNAP hairpin U-stretch transcription terminator. Single-stranded nucleic acids are shown to bind more tightly than double-stranded nucleic acids to T7 RNAP. These observations and a large set of published studies on T7 RNAP structure and mechanism are accommodated in a relatively simple model of T7 RNAP transcription initiation and termination in which a T7 RNAP that has initiated transcription is proposed to be capable of assuming two functionally distinct conformations: an abortive conformer characterized by a loose association with the nascent RNA and an inability to translocate along the template; and a processive conformer characterized by the stable retention of the nascent RNA and the ability to process stably along the template. The equilibrium between these two conformations is shifted towards the processive form when the nascent chain binds at a site located at least partly on the T7 RNAP N-terminal domain. The interaction requires that the RNA be more than approximately nine bases and this RNAP-RNA interaction plays a primary role in retaining the RNA within the ternary complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Single crystals of a chimeric T7/T3 RNA polymerase with T3 promoter specificity and a nonprocessive T7 RNAP mutant.

Two RNA polymerases homologous to bacteriophage T7 RNA polymerase, bacteriophage Sp6 and T3 RNA polymerases, were screened for crystallization under conditions identical or similar to those reported for the growth of large single crystals of T7 RNA polymerase (Sousa, R., Rose, J. P., Chung, Y. J., Lafer, E. M., and Wang, B.-C. (1989) Proteins 5, 266; Sousa, R., Lafer, E. M., and Wang, B.-C. (1990) J. Crystal Growth, in press; Sousa, R., and Lafer, E. M. (1990) Methods 1, in press). A number of mutant T7 RNAPs were also screened under these conditions as were three chimeric RNA polymerases consisting of T7 RNAP N-terminal and T3 RNAP C-terminal sequences. One chimeric polymerase and two mutant polymerases crystallized readily under T7 RNAP crystallization conditions. The chimeric polymerase crystallized in a space group different from T7 RNA polymerase: orthorombic with unit cell parameters a = 75 A, b = 98 A, c = 159 A; space group P2(1)2(1)2(1) and 4 molecules/unit cell. This chimeric enzyme exhibits T3 promoter specificity and will make it possible to investigate how structural differences between the T3 and T7 RNA polymerase promoter recognition domains determine their different promoter specificities. One of the mutant polymerases successfully crystallized was an enzyme which can carry out promoter recognition and abortive transcription but cannot carry out processive transcription. Its structure may provide information on the nature of the conformational changes undergone by T7 RNAP in the abortive-processive switch. Crystals of the second mutant T7 RNA polymerase were unsuitable for x-ray analysis.