Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.
Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.
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真核翻译起始因子 eIF5 通过调节 Pi 释放和前起始复合物的构象转变来提高起始密码子识别的准确性。
DOI：
10.1093/nar/gkv510
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发表时间：
2015
影响因子：
14.9
通讯作者：
Hinnebusch,AlanG
中科院分区：
文献类型：
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作者：
Saini,AdeshK;Nanda,JagpreetS;Martin-Marcos,Pilar;Dong,Jinsheng;Zhang,Fan;Bhardwaj,Monika;Lorsch,JonR;Hinnebusch,AlanG
图2. TIF 5等位基因SUI 5/G31 R的基因内抑制基因的遗传表征。(A)his 4 -301 tif 5 Δ菌株ASY 100的衍生物，携带TIF 5 + URA 3质粒和LEU 2质粒上指定的TIF 5-FL等位基因（hc WT对pAS 5 -132，sc WT对pAS 5 -101，G31 R对pAS 5 -111，G31 R，M18 V对pAS 5 -115，G31 R，K33 E对pAS 5 -117，G31 R，G62 S对pAS 5 -112，G31 R，将pAS 5 -116上的hc L 61 A和pAS 5 -136上的hc G31 R、T34 N）复制平板接种至补充有0.3 mM组氨酸（-LU）或0.0003 mM组氨酸（-LUH）的SC-LU或补充有5.2 mM 5-FOA（5-FOA）的SC-L。将细胞在30 ℃下孵育3天（-LU）或6天（-LUH和5-FOA）。(B)将（A）中所述菌株的10倍系列稀释液点在SC-LU上，并在30 ℃下孵育3天。(C)用具有AUG（p367）或UUG（p391）起始密码子的HIS 4-lacZ报告质粒转化携带TIF 5 + TRP 1质粒和LEU 2质粒上指示的TIF 5等位基因的tif 5 Δ菌株ASY 137的衍生物。将细胞在30 ° C下在缺乏亮氨酸、色氨酸和尿嘧啶的SC中培养，并在全细胞提取物（WCE）中测量β-半乳糖苷酶活性。从三个独立的转化体计算从UUG表达的β-半乳糖苷酶与AUG报告基因的比率，并绘制平均比率和SEM（误差条）。(D)携带（A）中所述TIF 5等位基因的his 4 -301 tif 5 Δ菌株的Slg−和His+/Sui−表型，以及在pAS 5 -106上含有M18 V、在pAS 5 -108上含有K33 E、在pAS 5 -103上含有G62 S、在pAS 5 -107上含有L 61 A或在pAS 5 -135上含有hc T34 N的等基因菌株，通过在补充有0.3 mM His（+ His）或0.0003 mM His（-His）的SC-LU上点样连续10倍稀释液并在30 ℃下孵育3 d（-His）或6 d（+ His）来测定。(E)用AUG或UUG HIS 4-lacZ报告质粒转化his 4 -301 sui 1 Δ菌株ASY 250的衍生物，其中染色体TIF 5基因在GAL 1启动子（PGAL-TIF 5）下，在TRP 1质粒上携带SUI 1+，在LEU 2质粒上携带指定的TIF 5等位基因。将转化体在具有2%半乳糖作为碳源并补充有0.3mM组氨酸（SGal+ H）的合成基本培养基中培养，然后转移到具有2%葡萄糖（SD+ H）的合成基本培养基中培养16小时。HIS 4-lacZ报告基因的UUG：AUG起始比如（C）中所述测定。(F)对于（D）中所述的携带所示质粒携带的TIF 5等位基因的菌株，如（C）中所述测量HIS 4-lacZ UUG：AUC起始比率。对于图A和D，图像已从在相同实验中平行检查的不同板获得的结果中裁剪。
Figure 2. Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R.(A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R, M18V on pAS5-115, G31R, K33E on pAS5-117, G31R, G62S on pAS5-112, G31R, L61A on pAS5-116 and hc G31R, T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30◦ C.(B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30◦ C.(C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30◦ C and ß-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of ß-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and SEMs (error bars) were plotted.(D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+ His) or 0.0003 mM His (− His) and incubated for 3d (− His) or 6d (+ His) at 30◦ C.(E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+ H) and then shifted to synthetic minimal medium with 2% glucose (SD+ H) for 16 h. UUG: AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C).(F) HIS4-lacZ UUG: AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.