A coordinated proteomic approach for identifying proteins that interact with the E. coli ribosomal protein S12.
A coordinated proteomic approach for identifying proteins that interact with the E. coli ribosomal protein S12.
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一种协调的蛋白质组学方法，用于鉴定与大肠杆菌核糖体蛋白S12相互作用的蛋白质。
DOI：
10.1021/pr3009435
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发表时间：
2013-03-01
影响因子：
4.4
通讯作者：
Kowalak JA
中科院分区：
文献类型：
--
作者：
Strader MB;Hervey WJ 4th;Costantino N;Fujigaki S;Chen CY;Akal-Strader A;Ihunnah CA;Makusky AJ;Court DL;Markey SP;Kowalak JA
细菌核糖体蛋白S12在环区含有一个普遍保守的D88残基，由于其位于30S亚基的a位点附近，因此被认为在翻译过程中起关键作用。虽然D88突变体是致命的，但已经发现该残基被翻译后修饰为β-甲基硫代天冬氨酸，这是一种翻译后修饰（PTM），在几种系统发育不同的细菌的S12同源物中发现。在之前的一篇报道中，我们的研究结果表明，这个保守的环区可能参与了蛋白质-蛋白质相互作用的形成。为了继续这项研究，我们利用两步互补亲和纯化策略对含有D88的环进行了探测，以鉴定候选结合物。第一个涉及内源性表达的S12蛋白，该蛋白含有用于捕获S12结合伴侣的c末端标签。第二种策略利用合成的生物素化肽代表D88保守环区来捕获S12环相互作用伙伴。利用SDS-PAGE和一维液相色谱串联质谱法对两种方法捕获的蛋白质进行检测。本报告的结果揭示了与30S亚基形成直接相互作用的蛋白，并阐明了可能与S12相互作用的蛋白。此外，我们提供的证据表明，在胁迫条件下参与调节核糖体和/或mRNA转录水平的两种蛋白质，RNase R和Hfq，与S12保守环形成直接相互作用，这表明它可能是蛋白质结合界面的一部分。
The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming proteins-protein interactions. To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop suggesting that it is likely part of a protein binding interface.
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影响因子：
3.2
作者：
Carr, JF;Hamburg, DM;Dahlberg, AE
通讯作者：
Dahlberg, AE
影响因子：
64.8
作者：
MIZUSHIMA, S;NOMURA, M
通讯作者：
NOMURA, M
影响因子：
3.2
作者：
Gustafsson, C;Persson, BC
通讯作者：
Persson, BC
DOI：
10.1073/pnas.0708608105
发表时间：
2008-02-12
影响因子：
11.1
作者：
Anton, Brian P.;Saleh, Lana;Roberts, Richard J.
通讯作者：
Roberts, Richard J.
影响因子：
7
作者：
Gupta, Nitin;Tanner, Stephen;Pevzner, Pavel A.
通讯作者：
Pevzner, Pavel A.