• 미생물학회지
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• 제27권4호
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• pp.317-322
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• 1989

A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

• 한국균학회지
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• 제49권3호
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• pp.285-294
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• 2021

In general, mycoviruses remain latent and rarely cause visible symptoms in fungal hosts; however, some viral infections have demonstrated abnormal mycelial growth and fruiting body development in commercial macrofungi, including Lentinula edodes. Compared to other cultivated mushrooms, L. edodes is more vulnerable to viral infections as it is still widely cultivated under near-natural conditions. In this study, we investigated whether Korean wild strains of L. edodes were infected by RNA mycoviruses that have previously been reported in other parts of the world (LeSV, LePV1, LeV-HKB, LeNSRV1, and LeNSRV2). Using specific primer sets that target the RNA-dependent RNA polymerase genes of each of the RNA mycovirus, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect viral infection. Viral infection was detected in about 90% of the 112 wild strains that were collected in Korea between 1983 and 2020. Moreover, multiple infections with RNA mycoviruses were detected in strains that had normal fruiting bodies. This work contributes to our understanding of the distribution of RNA mycoviruses in Korea and the impact of multiple viral infections in a single strain of L. edodes.

• Bulletin of the Korean Chemical Society
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• 제30권5호
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• pp.1039-1042
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• 2009

RNA polymerase II C-terminal domain phosphatase 1 containing ubiquitin like domain (UBLCP1) has been identified as a regulatory molecule of RNA polymerase II. UBLCP1 consists of ubiquitin like domain (UBL) and phosphatase domain homologous with UDP and CTD phosphatase. UBLCP1 was cloned into the E.coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using both affinity and gel-filtration chromatography. Domains of UBLCP1 protein were successfully purified as 7 mg/500 mL (UBLCP1, 36.78 KDa), 32 mg/500 mL (UBL, 9 KDa) and 8 mg/500 mL (phosphatase domain, 25 KDa) yielded in LB medium, respectively. Isotope-labeled samples including triple-labeled ($^2H/^{15}N/^{13}C$) UBLCP1 were also prepared for hetero-nuclear NMR experiments. $^{15}N-^{1}H$ 2D-HSQC spectra of UBLCP1 suggest that both UBL and phosphatase domain are properly folded and structurally independent each other. These data will promise us further structural investigation of UBLCP1 by NMR spectroscopy and/or X-ray crystallography.

• Journal of Microbiology and Biotechnology
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• 제4권3호
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• pp.183-190
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• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Journal of Microbiology
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• 제42권2호
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• pp.103-110
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• 2004

In order to understand the molecular basis of selective expression of stationary-phase genes by RNA polymerase containing$\sigma$$\^$38/ (E$\sigma$$\^$38/) in Escherichia coli, we examined transcription from the stationary-phase promoters, katEP, bo1AP, hdeABP, csgBAP, and mcbP, in vivo and in vitro. Although these pro-moters are preferentially recognized in vivo by E$\sigma$$\^$38/, they are transcribed in vitro by both E$\sigma$$\^$38/ and E$\sigma$$\^$70/ containing the major exponential $\sigma$, $\sigma$$\^$70/. In the presence of high concentrations of glutamate salts, how-ever, oldy E$\sigma$$\^$38/ was able to efficiently transcribe from these promoters, which supports the concept that the promoter selectivity of $\sigma$$\^$38/-containing RNA polymerase is observed only under specific reaction con-ditions. The examination of 6S RNA, which is encoded by the ssr1 gene in vivo, showed that it reduced E$\sigma$$\^$70/ activity during the stationary phase, but this reduction of activity did not result in the elevation of E$\sigma$$\^$38/ activity. Thus, the preferential expression of stationary-phase genes by E$\sigma$$\^$38/ is unlikely the con-sequence of selective inhibition of E$\sigma$$\^$70/ by 6S RNA.

• Bulletin of the Korean Chemical Society
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• 제31권7호
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• pp.2107-2109
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• 2010

• BMB Reports
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• 제30권5호
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• pp.362-369
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• 1997

Multiple phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit in RNA polymerase II (RNAP II) is thought to play an important role in the transcription cycle. The preinitiation complex in a partially purified complete transcription system suggested that RNA polymerase IIA containing unphosphorylated CTD is involved in complex assembly, whereas RNA polymerase IIO containing Ser and Thr phosphorylated CTD is not involved in preinitiation complex assembly. Recently a minimal transcription system was developed which requires chemically defined minimal components for its transcription: TBP, TFIIB, TFIIF, RNAP II and a supercoiled adenovirus-2 major late promoter (Ad-2 MLP). It would be using interesting to determine the consequence of CTD phosphorylation on preinitiation complex formation using the minimal transcription system. Contrary to the results from the partially purified complete transcription system, both RNA polymerase IIA and IIO are equally recruited in the preinitiation complex formation. The discrepancy may result from the two different assays used to determine complex formation, the use of chemically undefined complete and defined minimal transcription systems. This implicates that some factors in the complete transcription system are involved in the distinction between RNAP IIA and IIO in complex assembly. In addition multiple tyrosine phosphorylation of the CTD of RNAP II was prepared with c-Abl kinase and its recruiting ability in the preinitiation complex was examined. Compare with Ser and Thr phosphorylated RNAP IIO, Tyr phosphorylated RNAP IlOy forms a stable preinitiation complex in both the minimal and complete transcription systems. Based on these results, it seems that tyrosine phosphorylation of the CTD is important in the transcription cycle on the special subset of class-II promoter or has a different role in the transcription process.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1744-1754
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• 2014

The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1634-1639
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• 2006

Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

• Bulletin of the Korean Chemical Society
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• 제34권3호
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• pp.837-850
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• 2013

Bovine viral diarrhea virus (BVDV), a major pathogen of cattle, is a well-characterized pestivirus which has been used as a good model virus for HCV. The RNA-dependent RNA polymerase (RdRp) plays a key role in the RNA replication process, thus it has been targeted for antivirus drugs. We employed two-dimensional quantitative structure-activity relationship (2D-QSAR) and molecular field analysis (MFA) to identify the molecular substructure requirements, and the particular characteristics resulted in increased inhibitory activity for the known series of compounds to act as effective BVDV inhibitors. The 2D-QSAR study provided the rationale concept for changes in the structure to have more potent analogs focused on the class of arylazoenamines, benzimidazoles, and acridine derivatives with an optimal subset of descriptors, which have significantly contributed to overall anti-BVDV activity. MFA represented the molecular patterns responsible for the actions of antiviral compound at their receptors. We conclude that the polarity and the polarizability of a molecule play a main role in the inhibitory activity of BVDV inhibitors in the QSAR modeling.