Christie G E, Calendar R
J Mol Biol. 1985 Feb 5;181(3):373-82. doi: 10.1016/0022-2836(85)90226-8.
The late genes of bacteriophage P2 are clustered into four transcription units. We have reported the transcription initiation sites for two of the late messenger RNAs, encoding genes QP and ONMLKRS. We have now located the 5' ends of the two remaining late mRNAs. The first gene in the VJHG transcription unit has been located by DNA sequence determination of the single nucleotide change in a V amber mutant. Location of the first gene in the FETUD transcription unit has been inferred from the DNA sequence. The 5' ends of the mRNAs for these two transcription units were located by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S1. Similar protection of hybrids using RNA that had been 5' end-labeled with [alpha-32P]GTP and guanylyl transferase confirmed that these 5' termini resulted from initiation of transcription. The DNA sequences preceding the P2 late transcription starts are different from the Escherichia coli promoter consensus sequences at -10 and -35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The four P2 late promoters do share sequence homologies in the -10 and -35 regions, however, and several additional homologies further upstream. P2 late gene expression also appears to involve negative regulation by a product of the ONMLKRS gene cluster. When cells are infected with P2 polar O amber mutants, a marked increase in the levels of proteins encoded by the other three gene clusters is observed. This increase is reflected in the amounts of late mRNAs, suggesting that RNA synthesis is normally repressed or that late mRNAs are more labile in the presence of a gene product from the ONMLKRS transcription unit. Satellite phage P4 induced P2 late gene expression without the usual requirement for P2 DNA replication. The 5' ends of the P2 late mRNAs are the same during P4 transactivation as during normal P2 late gene expression. Thus, the regulation of P2 late gene expression by P4 does not involve altered promoter selection.
噬菌体P2的晚期基因聚集成四个转录单元。我们已经报道了两个晚期信使RNA的转录起始位点，它们分别编码基因QP和ONMLKRS。我们现在已经确定了另外两个晚期mRNA的5'端。通过对V琥珀突变体中单个核苷酸变化的DNA序列测定，确定了VJHG转录单元中的第一个基因。FETUD转录单元中第一个基因的位置是根据DNA序列推断出来的。通过核酸酶S1消化RNA-DNA杂交体中末端标记的限制片段来确定这两个转录单元mRNA的5'端。使用用[α-32P]GTP和鸟苷酸转移酶进行5'端标记的RNA对杂交体进行类似的保护，证实这些5'末端是转录起始的结果。P2晚期转录起始前的DNA序列在-10和-35处与大肠杆菌启动子共有序列不同，这与P2晚期基因表达调控中明显需要噬菌体编码的蛋白质一致。然而，四个P2晚期启动子在-10和-35区域确实共享序列同源性，并且在更上游还有几个额外的同源性。P2晚期基因表达似乎也涉及ONMLKRS基因簇产物的负调控。当细胞用P2极性O琥珀突变体感染时，观察到其他三个基因簇编码的蛋白质水平显著增加。这种增加反映在晚期mRNA的量上，表明RNA合成通常受到抑制，或者在存在来自ONMLKRS转录单元的基因产物时晚期mRNA更不稳定。卫星噬菌体P4在没有通常对P2 DNA复制的要求的情况下诱导P2晚期基因表达。在P4反式激活期间，P2晚期mRNA的5'端与正常P2晚期基因表达期间相同。因此，P4对P2晚期基因表达的调控不涉及启动子选择的改变。
