Temporal regulation of gene expression of the Thermus thermophilus bacteriophage P23-45.
Temporal regulation of gene expression of the Thermus thermophilus bacteriophage P23-45.
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DOI：
10.1016/j.jmb.2010.10.049
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发表时间：
2011-01-07
影响因子：
5.6
通讯作者：
Minakhin L
中科院分区：
文献类型：
--
作者：
Berdygulova Z;Westblade LF;Florens L;Koonin EV;Chait BT;Ramanculov E;Washburn MP;Darst SA;Severinov K;Minakhin L
嗜热菌Thermus thermophilus(T.th.)感染过程中基因表达的调控用噬菌体P23-45对HB8进行了研究。大芯片分析揭示了宿主转录的关闭，并鉴定了三个时间类的噬菌体基因：早期、中期和晚期。引物延伸实验表明，P23-45早期转录本的5‘端之前有一个共同的序列基序，可能定义了早期病毒启动子。T.HB8RNA聚合酶(RNAP)在体外识别中、晚期噬菌体启动子，但不识别早期启动子。体内实验显示，在感染细胞中存在对利福平耐药的RNA聚合活性，负责早期转录。P23-45早期基因的产物与多亚基RNAPs的最大催化亚基有较远的序列相似性，并含有保守的金属结合基序，是这些蛋白质的诊断基序。我们推测ORF64编码识别早期噬菌体启动子的抗利福平噬菌体RNAP。T.Th.的亲和分离来自P23-45感染细胞的HB8 RNAP发现了两种噬菌体编码蛋白：gp39和gp76，它们与宿主RNAP结合并抑制宿主启动子的体外转录，但不抑制中晚期噬菌体启动子的转录，因此可能控制感染过程中宿主基因向病毒基因表达的转变。据我们所知，gp39和gp76是第一个由嗜热噬菌体编码的细菌RNAP结合蛋白。
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.
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