Bacteriophage P22 antitermination boxB sequence requirements are complex and overlap with those of λ
Bacteriophage P22 antitermination boxB sequence requirements are complex and overlap with those of λ
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DOI：
10.1128/jb.00059-08
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发表时间：
2008-06-01
影响因子：
3.2
通讯作者：
Smith, Colin A.
中科院分区：
文献类型：
--
作者：
Cocozaki, Alexis I.;Ghattas, Ingrid R.;Smith, Colin A.
在噬菌体lambda和P22中，转录抗终止使用N蛋白结合在调控转录物中类似的boxB RNA发夹。与λ N-boxB相互作用相反，P22 N-boxB相互作用尚未被广泛研究。P22 N肽boxB（左）复合体的核磁共振结构和有限的突变已被报道，但没有揭示boxB的一致序列。我们使用了一种基于质粒的抗终止系统来筛选随机循环的boxB和测试boxB突变体。我们发现P22 N要求boxB具有一个类似gnra的环路，而对环路或干中的剩余序列没有简单的要求。U:A或A:U碱基对强烈倾向于靠近环，并且似乎与环和远端茎合作调节N结合。一些类似gnra的六环具有适度的活性。一些boxB突变体结合P22和lambda N，表明P22 N对boxB的要求与lambda N的要求重叠。点突变可以显著改变P22和lambda N之间的boxB特异性。P22 N特异性的boxB可以通过一系列单突变通过双功能中间体突变为lambda N特异性，正如中性进化理论所预测的那样。
Transcription antitermination in phages lambda and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the lambda N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB(left) complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and lambda N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by lambda N. Point mutations can dramatically alter boxB specificity between P22 and lambda N. A boxB specific for P22 N can be mutated to lambda N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.