Molecular Basis of RNA: Protein Recognition by the MiaA tRNAPrenyl Transferase of E. coli

;​R o o t E n t y F 嗯 C o m p o b j b W o r d d o c u m e n t u O b j e c t P O O l 我我A, b, c, d， I, jF Microsoft Word 6.0文档MSWordDoc。6;​‘, R 9 @ D ’ ' G c 3 q G -；PRIVATE本提案的总体目标是研究一类重要的tRNA修饰酶- tRNA戊烯基转移酶-识别RNA：蛋白质的分子基础。作为实验模型系统，我们将分析大肠杆菌k12miaa tRNA戊烯基转移酶。MiaA酶在46种大肠杆菌tRNA中8种的37位反密码子旁的腺苷N6氮上催化二磷酸二甲基丙烯基（DMADP）的2异戊烯基（即i6a37形成）。MiaA戊烯基转移酶将被过表达和纯化到均匀性，可能借助于可移动的亲和力标签。将建立MiaA酶形成i6a37所需的组合tRNA序列和二级结构决定因子。最后，将确定MiaA酶中四个高度保守的氨基酸基序在底物结合和i6a37形成中的作用。这些综合数据将使我们更全面地了解细胞如何调节tRNA修饰水平，并在不同生理条件下协调tRNA修饰与tRNA水平的变化。最后，或许也是最重要的一点，提出的实验可能会带来RNA一般问题的新概念：蛋白质识别。MiaA中没有一个保守的蛋白基序与已知的RNA或DMADP结合位点强烈匹配，一个基序与ATP/GTP结合位点相对应，尽管已知i6a37的形成并不需要三磷酸核苷酸。几个假设将测试这些保守基序在MiaA酶功能中的作用。RNA是一种大的核酸分子，在所有生物体的细胞中起着许多重要的生物学作用。为了发挥作用，不同种类的RNA与蛋白质特异性地相互作用，蛋白质在细胞中充当酶催化剂和结构成分。因此，了解不同种类的RNA识别特定蛋白质的原理具有重要的生物学意义。迄今为止，这些原则仅适用于少数RNA与蛋白质的相互作用，尚未出现共识规则。这项NSF拨款的总体目标是确定一种重要的模式细菌蛋白质的RNA：蛋白质识别原理，称为MiaA戊烯基转移酶，它特异性地结合并改变某些转移RNA （tRNA）分子的碱基（即修饰）。通过将结构变化与功能特性联系起来，应该有可能找出tRNA：蛋白质识别这些重要大分子的规则。*** ;@ ....（）（）) ()() ()() Z：凤凰W CH o；如果我是你，我是你，我是你，我是你，我是你Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT marcia steinberg marcia steinberg @ @ @ UZ @ Microsoft Word 6.0 3e3eu1C L N N N 1

; R o o t E n t r y F mhm C o m p O b j b W o r d D o c u m e n t u O b j e c t P o o l i i A B C D E F G H I J F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; ' , R 9 @ D ` ' G c 3 q G - ; v ' b N c 9420416 Winkler PRIVATE The overall goal of this proposal is to investigate the molecular basis for RNA:protein recognition by an important class of tRNA modification enzymes, the tRNA prenyl transferases. As an experimental model system, the E. coli K 12 MiaA tRNA prenyl transferase will be analyzed. The MiaA enzyme catalyzes the addition of a (2 isopentenyl group from dimethylallyl diphosphate (DMADP) to the N6 nitrogen of adenosine adjacent to the anticodon at position 37 of 8 of 46 E. coli tRNA species (i.e., i6A 37 formation). The MiaA prenyl transferase will be overexpressed and purified to homogeneity, possibly with the aid of removable affinity tags. The combined tRNA sequence and secondary structure determinants required by the MiaA enzyme for i6A 37 formation will be established. Finally, the roles of four highly conserved amino acid motifs in the MiaA enzyme will be ascertained for substrate binding and i6A 37 formation. These combined data should lead to a more complete picture of how cells regulate tRNA modification levels and coordinate modification with changes in tRNA levels unde r different physiological conditions. Finally, and perhaps most importantly, the proposed experiments may lead to new concepts in the general problem of RNA:protein recognition. None of the conserved protein motifs in MiaA strongly matches known RNA or DMADP binding sites, and one motif corresponds to an ATP/GTP binding site, even though nucleotide triphosphates are not known to be required for i6A 37 formation. Several hypotheses will be tested for the role of these conserved motifs in MiaA enzyme function. %%% RNA is a large nucleic acid molecule that plays numerous essential biological roles in the cells of all organisms. To function, different kinds of RNA interact specifically with proteins, which act as enzyme catalysts and structural components in cells. Thus, it is of fundamental biological importance to learn the principles by which different kinds of RNA recognize specific proteins. To date, these principles are known for only a small number of RNA:protein interactions, and no consensus rules have emerged. The overall goal of this NSF grant is to determine the principles of RNA:protein recognition for an important model bacterial protein, called the MiaA prenyl transferase, that specifically binds to and changes bases of (i.e., modifies) certain transfer RNA (tRNA) molecules.. By correlating structural changes with functional properties, it should be possible to work out the rules for tRNA:protein recognition for these important kinds of macromolecules. *** ; @ ....()()))()() Z : phoenix W CH o ; S u m m a r y I n f o r m a t i o n ( @ Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ @ @ UZ @ Microsoft Word 6.0 3 e 3 e u 1 C L N N N l