Austin Ryan J, Xia Tianbing, Ren Jinsong, Takahashi Terry T, Roberts Richard W
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Biochemistry. 2003 Dec 23;42(50):14957-67. doi: 10.1021/bi0351312.
N proteins from bacteriophages lambda, P22, and phi21 modulate transcription elongation by binding nascent "boxB" mRNA hairpins. This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets. Here, we have analyzed the affinity and specificity of the peptide-RNA interactions that modulate this transcriptional switch. To do this, we constructed a series of peptides based on the wild-type lambda, P22, and phi21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage. Binding constant (K(d)) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added. K(d)'s demonstrate that lambda and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins. Surprisingly, phi21 shows very little specificity for its cognate targets. Lambda and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively. We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity. Amino acid coupling analysis supports a binding model where the Arg8 residue of lambda peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.
来自噬菌体λ、P22和φ21的N蛋白通过结合新生的“boxB”mRNA发夹结构来调节转录延伸。这种RNA识别是由能够与其同源boxB RNA靶标相互作用的富含N端精氨酸的肽序列介导的。在这里，我们分析了调节这种转录开关的肽-RNA相互作用的亲和力和特异性。为此，我们基于野生型λ、P22和φ21的N蛋白结合结构域构建了一系列长度从11到22个残基的肽，并分析了它们与这三种噬菌体的向左和向右boxB RNA发夹靶标的相互作用。使用用2-氨基嘌呤（2AP）标记的RNA发夹并监测添加肽时的荧光变化来确定结合常数（K(d)）值。K(d)值表明，λ和P22的N肽以高特异性结合其同源boxB靶标，并且对其向左和向右的发夹表现出相同的亲和力。令人惊讶的是，φ21对其同源靶标几乎没有特异性。λ和P22的N肽分别通过其氨基末端和羧基末端模块赋予的特异性表现出不同的识别模式。我们生成了一个取代肽的相互矩阵，以检查单个残基对特异性的贡献。氨基酸偶联分析支持一种结合模型，其中λ肽的Arg⁸残基作为一个构象热点，锚定其boxB发夹靶标的诱导环折叠。
