Functional characterization of the ribosomal tunnel exit ligand ERj1

ERj1是内质网（ER）的膜驻留Hsp40伴侣子，它通过内质网膜招募ER腔Hsp70伴侣子BiP来翻译核糖体。此外，ERj1在没有BiP的情况下通过阻断起始来调节核糖体的翻译活性。此外，ERj1被认为是潜在的转录因子。我们在ERj1的胞质结构域中发现了一个非肽，该非肽负责核糖体相互作用，并将其定性为翻译活性的调节剂。在核糖体水平上，rRNA参与了ERj1的结合，并通过生物化学和低温电镜技术确定了核糖体的出口位点为相互作用位点。因此，翻译的调节涉及一种变构机制，显然是由核糖体的构象变化介导的。由于ERj1同时与核糖体和BiP结合，因此它注定会参与蛋白质转运到内质网的过程。此外，我们假设ER1在基因表达调控中起着双重作用——在转录水平和翻译水平。我们项目的目的是最终在原子级别上阐明ERj1的功能。在这些研究中，与其他核糖体配体（NAC， RAC， Sec61复合物）进行比较的定量方面也将得到解决。

ERj1 is a membrane-resident Hsp40 chaperon of the endoplasmic reticulum (ER), which recruits the ER-lumenal Hsp70 chaperon, BiP via the ER membrane to translating ribosomes. Furthermore, ERj1 modulates the translational activity of ribosomes by blocking initiation in the absence of BiP. In addition, ERj1 was characterized as potential transcription factor. We identified a nonapeptide within the cytosolic domain of ERj1 as responsible for ribosome interaction and characterized it as the modulator of translation activity. At the level of the ribosome, rRNA was shown to be involved in binding of ERj1 and the exit site of the ribosome was identified as the interacting site by biochemistry and Cryo-EM. Thus, the modulation of translation involves an allosteric mechanism, apparently mediated by conformational changes in the ribosome. Since ERj1 simultaneously binds to both, the ribosome and BiP, it is predestined to be involved in the process of protein transport into the ER. Furthermore, we postulate that ER1 plays a dual role in the regulation of gene expression – at the level of transcription as well as translation. The aim of our project is to elucidate the function(s) of ERj1, ultimately at the atomic level. In these studies quantitative aspects in comparison to the other ribosomal ligands (NAC, RAC, Sec61 complex) will also be addressed.