Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability.

We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible "thumb" domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex.

Characterization of an unusual, sequence-specific termination signal for T7 RNA polymerase.

We have characterized an unusual type of termination signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the non-template strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.

Z-band proteins in the flight muscle and leg muscle of the honeybee.

Monoclonal antibodies (mAb's) have been raised against proteins in preparations of Z-discs isolated from honeybee fibrillar flight muscle. These antibodies have identified four Z-disc antigens on immunoblots of honeybee fibrillar proteins. Antibody alpha binds to the 90-100 kD protein, alpha-actinin; mAb P interacts with the protein, projectin, an extremely large polypeptide (greater than 600kD) found in the connecting filaments which link thick filaments to the Z-band in insect asynchronous flight muscle. Two other mAb's recognize previously uncharacterized insect Z-band proteins. Monoclonal antibody Z(400) binds to a pair of proteins with molecular masses near 400 kD and 600 kD. Antibody Z(175) recognizes two components, 158 kD and 175 kD, that are not only immunologically similar but have nearly identical peptide maps. Indirect immunofluorescence microscopy studies show that the proteins recognized by mAb's alpha, Z(175) and Z(400) are located at the Z-band, while the mAb P antigen is found on either side of it. Three of the four antibodies we have obtained recognize leg muscle proteins. Monoclonal antibodies alpha and P comigrate on SDS gels with analogous components from flight muscle. Only the smaller of the two proteins identified in flight muscle by mAb Z(400) is found in leg muscle, however. Furthermore, no Z(175) antigens have been detected in the non-fibrillar tissue by either monoclonal or polyclonal antibodies. Immunofluorescence microscopy studies localize the alpha and Z(400) antigens at the Z-line in leg muscle fibrils. Surprisingly, however, mAb P binds within the A-bands of synchronous fibres, not between the A- and Z-bands as in asynchronous fibrillar muscle.

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

Interferon and cis-DDP: combination chemotherapy for P388 leukemia in CDF1 mice.

CDF1 mice were implanted with 10(6) P388 leukemia cells. One day postimplantation, when interferon (IFN) therapy was begun, the tumor burden was ca. 4.8 X 10(6) cells. Therapy consisted of nine consecutive daily intraperitoneal (i.p.) injections of 10(6) units of Ehrlich ascites tumor cell IFN (specific activity 5 X 10(7) IU/mg). The median life span for control animals was 10.0 days versus 18.5 days for the treated animals. It was estimated that about 2 X 10(6) viable P388 cells were present on Day 9 when therapy was discontinued. There was no evidence of toxicity in animals receiving 10(6) units of IFN/day for nine consecutive days. In a separate experiment, control animals challenged as above had a median life span of 9.5 days of versus 15 days for animals receiving 10(6) units of IFN qd on Days 2-10. A third group of animals receiving a single injection of cis-DDP on Day 1 had a median survival of 18.5 days, while a fourth group receiving both cis-DDP on Day 1 and subsequent treatment with IFN survived 26 days. The results are discussed in terms of impact on tumor cell population.