TEMPORAL REGULATION OF GENE EXPRESSION OF TTHERMOPHILUS BACTERIOPHAGE P23-45

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 嗜热菌感染过程中基因表达的调控 Thermus thermophilus（T.（第10页）研究了HB 8与噬菌体P23-45的作用。 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 ′端之前有一个 可能定义早期病毒启动子的共同序列基序。T.日HB8 RNA RNAP在体外识别噬菌体中期和晚期启动子， 不承认早期的推动者。体内实验表明， 感染细胞中利福平耐药RNA聚合活性 早期转录P23-45早期基因64的产物显示了一个远距离表达。 与多亚基RNAP的最大催化亚基的序列相似性， 含有保守的金属结合基序，是这些蛋白质的诊断。我们 假设ORF 64编码利福平抗性噬菌体RNAP， 早期噬菌体启动子亲和分离T.日来自P23-45感染的HB 8 RNAP 细胞鉴定出两种噬菌体编码的蛋白质：gp 39和gp 76，它们与宿主结合 RNAP和抑制宿主启动子的体外转录，但不抑制中间启动子的转录。 或晚期噬菌体启动子，因此可以控制从宿主到病毒基因的转移 感染时的表达。据我们所知，gp 39和gp 76是第一个 由嗜热噬菌体编码的特征性细菌RNAP结合蛋白。

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.