TEMPORAL REGULATION OF GENE EXPRESSION OF TTHERMOPHILUS BACTERIOPHAGE P23-45

嗜热菌噬菌体 P23-45 基因表达的时间调控

• 批准号: 8361589
• 负责人: Leonid Minakhin
• 金额: $ 0.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361589
• 关键词: Affinity; Bacterial RNA; Bacteriophages; Binding; Binding Proteins; CD40 Ligand; Catalytic Domain; Cells; DNA-Directed RNA Polymerase; Diagnostic; Distant; Early Promoters; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Grant; In Vitro; Infection; Metals; National Center for Research Resources; Primer Extension; Principal Investigator; Proteins; RNA; Research; Research Infrastructure; Resources; Rifampicin resistance; Source; Thermus thermophilus; Transcript; United States National Institutes of Health; Viral; Viral Genes; cost; in vivo; macromolecule; promoter; research study; thermophilic bacteria

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Regulation of gene expression during infection of the thermophilic bacterium Thermus thermophilus (T. th.) HB8 with the bacteriophage P23-45 was investigated. Macroarray analysis revealed host transcription shut-off and identified three temporal classes of phage genes: early, middle, and late. Primer extension experiments revealed that the 5¿ ends of P23-45 early transcripts are preceded by a common sequence motif that likely defines early viral promoters. T. th. HB8 RNA polymerase (RNAP) recognizes middle and late phage promoters in vitro but does not recognize early promoters. In vivo experiments revealed the presence of rifampicin-resistant RNA polymerizing activity in infected cells responsible for early transcription. The product of the P23-45 early gene 64 shows a distant sequence similarity with the largest, catalytic subunits of multisubunit RNAPs and contains the conserved metal-binding motif that is diagnostic of these proteins. We hypothesize that ORF64 encodes rifampicin-resistant phage RNAP that recognizes early phage promoters. Affinity isolation of T. th. HB8 RNAP from P23-45-infected cells identified two phage-encoded proteins: gp39 and gp76, that bind the host RNAP and inhibit in vitro transcription from host promoters, but not from middle or late phage promoters, and may thus control the shift from host to viral gene expression during infection. To our knowledge, gp39 and gp76 are the first characterized bacterial RNAP-binding proteins encoded by a thermophilic phage.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 嗜热细菌感染过程中基因表达的调控 研究了带有噬菌体 P23-45 的嗜热栖热菌 (T.th.) HB8。 宏阵列分析揭示了宿主转录关闭并确定了三个 噬菌体基因的时间类别：早期、中期和晚期。引物延伸 实验表明，P23-45 早期转录本的 5¿ 末端前面有一个 可能定义早期病毒启动子的共同序列基序。 T.th。 HB8 RNA 聚合酶（RNAP）在体外识别中晚期噬菌体启动子，但不识别中晚期噬菌体启动子。 不认识早期启动子。体内实验表明存在 感染细胞中耐利福平的 RNA 聚合活性负责 早期转录。 P23-45早期基因64的产物显示出遥远的 与多亚基 RNAP 的最大催化亚基的序列相似性 含有可诊断这些蛋白质的保守金属结合基序。我们 假设 ORF64 编码耐利福平噬菌体 RNAP，该 RNAP 识别 早期噬菌体启动子。 T.th 的亲和分离来自 P23-45 感染的 HB8 RNAP 细胞识别出两种噬菌体编码的蛋白质：gp39 和 gp76，它们与宿主结合 RNAP 并抑制来自宿主启动子的体外转录，但不抑制来自中间体的转录 或晚期噬菌体启动子，因此可以控制从宿主基因到病毒基因的转变 感染期间的表达。据我们所知，gp39和gp76是第一个 表征了由嗜热噬菌体编码的细菌 RNAP 结合蛋白。