Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

原核表达系统T7RNA聚合酶/启动子在真核细胞中表达目的基因的实验研究

将目前高表达水平强大的原核表达系统之一T7 RNA聚合酶/启动子表达系统通过一系列改进引入真核细胞.通过转染真核细胞实验表明,采用真核启动子CMV调控T7 RNA聚合酶的表达和在T7启动子下游插入EMCV IRES序列两种解决方案能使该原核表达系统在真核细胞高效表达目的基因,且能适应不同的真核细胞环境,是一良好的细胞类型非依赖的表达体系.     

Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.     

RNA polymerases B and C are more closely related to each other than to RNA polymerase A

Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements.     

Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity

Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).     

Human cell lines expressing hormone regulated T7 RNA polymerase localized at distinct intranuclear sites

Although several systems are now available for the controlled expression of eukaryotic genes transcribed by RNA polymerase II, regulated expression has been more difficult to achieve in the case of genes transcribed by RNA polymerase III. In the present study the gene for bacteriophage T7 RNA polymerase, implanted with a eukaryotic nuclear localization signal, was linked to a 5'-flanking ecdysone-responsive promoter and stably transformed human cell lines were constructed in which the ecdysone promoter-T7 RNA polymerase gene had been integrated intact, as demonstrated by a polymerase chain reaction assay. Exposure of these cells to the ecdysone analog ponasterone A resulted in the appearance of a single protein having the expected size of T7 RNA polymerase in immunoblots of cell extracts probed with an affinity purified antibody raised against the C-terminus of T7 RNA polymerase. The induced T7 RNA polymerase was exclusively localized in the nucleus of induced cells and was undetectable in uninduced cells either by immunoblotting or immunofluorescence. The induced T7 RNA polymerase was present at numerous punctate foci dispersed throughout the nucleoplasmic regions of the nucleus and was also present in the nucleoli. Both of these observed intranuclear localizations have relevance to the potential applications of this system.