Department of Biology, Texas A&M University, College Station, Texas, USA.
Mol Cell Biol. 2012 Jul;32(13):2396-406. doi: 10.1128/MCB.00136-12. Epub 2012 Apr 16.
The fungal arginine attenuator peptide (AAP) is encoded by a regulatory upstream open reading frame (uORF). The AAP acts as a nascent peptide within the ribosome tunnel to stall translation in response to arginine (Arg). The effect of AAP and Arg on ribosome peptidyl transferase center (PTC) function was analyzed in Neurospora crassa and wheat germ translation extracts using the transfer of nascent AAP to puromycin as an assay. In the presence of a high concentration of Arg, the wild-type AAP inhibited PTC function, but a mutated AAP that lacked stalling activity did not. While AAP of wild-type length was most efficient at stalling ribosomes, based on primer extension inhibition (toeprint) assays and reporter synthesis assays, a window of inhibitory function spanning four residues was observed at the AAP's C terminus. The data indicate that inhibition of PTC function by the AAP in response to Arg is the basis for the AAP's function of stalling ribosomes at the uORF termination codon. Arg could interfere with PTC function by inhibiting peptidyltransferase activity and/or by restricting PTC A-site accessibility. The mode of PTC inhibition appears unusual because neither specific amino acids nor a specific nascent peptide chain length was required for AAP to inhibit PTC function.
真菌精氨酸衰减肽（AAP）由一个调节上游开放阅读框（uORF）编码。AAP 作为核糖体隧道内的新生肽，在响应精氨酸（Arg）时会使翻译停滞。在 Neurospora crassa 和小麦胚芽翻译提取物中，通过将新生 AAP 转移到嘌呤霉素上来分析 AAP 和 Arg 对核糖体肽基转移酶中心（PTC）功能的影响，作为一种测定方法。在高浓度 Arg 的存在下，野生型 AAP 抑制 PTC 功能，但缺乏停滞活性的突变型 AAP 则没有。虽然野生型长度的 AAP 最有效地使核糖体停滞，但根据引物延伸抑制（toeprint）测定和报告基因合成测定，在 AAP 的 C 末端观察到抑制功能的四残基窗口。数据表明，AAP 响应 Arg 抑制 PTC 功能是其在 uORF 终止密码子处使核糖体停滞的基础。Arg 可能通过抑制肽基转移酶活性和/或限制 PTC A 位的可及性来干扰 PTC 功能。PTC 抑制的模式似乎不寻常，因为 AAP 抑制 PTC 功能既不需要特定的氨基酸，也不需要特定的新生肽链长度。
