Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
RNA. 2014 Aug;20(8):1210-22. doi: 10.1261/rna.044529.114. Epub 2014 Jun 16.
Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.
大肠杆菌亮氨酰/苯丙氨酰-tRNA 蛋白转移酶催化在蛋白质多肽底物的 N 端将氨基酸通过 tRNA 进行翻译后添加。基于生化和结构研究，当前 L/F 转移酶的 tRNA 识别模型涉及 3' 氨酰腺苷的身份和氨酰-tRNA 的 D-茎与 L/F 转移酶上的正电荷簇的序列无关对接。然而，该模型无法解释 40 年前观察到的同功受体偏好。使用体外转录的 tRNA 和定量 MALDI-ToF MS 酶活性测定，我们已经证实，实际上，L/F 转移酶活性确实强烈偏爱最丰富的亮氨酰-tRNA，即 tRNA(Leu)（反密码子 5'-CAG-3'）同工受体。我们使用杂种 tRNA 构建物进一步研究了这种偏好的分子机制。我们在 tRNA(Leu)（CAG）的受体茎中鉴定出两个独立的序列元件-G₃:C₇₀ 碱基对和一组 4 个核苷酸（C₇₂、A₄:U₆₉、C₆₈）-这对于 L/F 转移酶的最佳结合和催化很重要。这映射了比以前提出的更具体、更依赖于序列的 L/F 转移酶 tRNA 识别模型。
