Molecular mechanism of translation arrest and mRNA degradation in cystathionine gamma-synthase, the key-step enzyme of methionine biosynthesis.

拟南芥胱硫醚γ-合酶（CGS）是催化甲硫氨酸生物合成的关键步骤。与生物合成途径中的许多关键步骤酶不同，CGS不是变构酶。CGS的表达在mRNA降解步骤中受到反馈调节，S-腺苷甲硫氨酸（SAM）充当效应器。mRNA降解后，形成5 '-截短的RNA物质作为降解中间体。位于CGS自身第一外显子编码区内的一段短的氨基酸，称为MTO 1区，在该调节中起顺式元件的作用。CGS调节发生在翻译过程中，并在小麦胚芽提取物的体外翻译系统中再现。使用体外系统的研究表明，在mRNA降解之前，SAM诱导在MTO 1区域周围的翻译延长停滞。因此，翻译延伸停滞引发mRNA降解。翻译停滞位点的精细定位显示，翻译停滞发生在位于MTO 1区域之后的Ser-94处，并且肽基-tRNA（Ser）积累。丙氨酸取代实验表明，除了MTO 1区氨基酸序列外，Trp-93和Ser-94对翻译停滞也很重要。新生肽通过核糖体出口通道从翻译核糖体中出现。该出口通道直径为15-20埃，长约100埃，新生肽的30-40个氨基酸残基将位于该通道中。MTO 1区域和阻滞位点（Ser-94）的相对位置表明，MTO 1区域新生肽位于出口通道内。对于CGS调控的遗传分析，分离了改变CGS表达的转录后调控的拟南芥突变体，并鉴定了它们的响应基因。

Cystathionine gamma-synthase (CGS) of Arabidopsis catalyzes the key step in methionine biosynthesis. Unlike many of the key-step enzymes in biosynthetic pathways, CGS is not an allosteric enzyme. Expression of CGS is feedback-regulated at the step of mRNA degradation and S-adenosylmethionine (SAM) acts as the effector. Upon mRNA degradation, 5'-truncated RNA species are formed as degradation intermediates. A short stretch of amino acids, termed the MTO1 region, located within the first exon-coding region of CGS itself, functions as the cis element in this regulation. CGS regulation occurs during translation and is reproduced in the in vitro translation system of wheat germ extract. Studies using the in vitro system revealed that, prior to the mRNA degradation, SAM induces translation elongation arrest at around the MTO1 region. Thus, translation elongation arrest triggers the mRNA degradation. Finer mapping of the translation arrest site showed that translation arrest occurs at Ser-94 located right after the MTO1 region, and peptidyl-tRNA(Ser) accumulates. Alanine substitution experiments identified that, in addition to the MTO1 region amino acid sequence, Trp-93 and Ser-94 are important for the translation arrest. A nascent peptide emerges from the translating ribosome through the ribosomal exit tunnel. This exit tunnel is 15-20 Angstroms in diameter and about 100 Angstroms long, and 30-40 amino acid residues of the nascent peptide will be located in the tunnel. Relative positions of the MTO1 region and the arrest site (Ser-94) suggest that the MTO1 region nascent peptide is located within the exit tunnel. For genetic analyses of the CGS regulation, Arabidopsis mutants that alter the post-transcriptional regulation of CGS expression were isolated and their responsive genes have been identified.