Holmberg L, Noller H F
Center for the Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, CA, 95064, USA.
J Mol Biol. 1999 Jun 4;289(2):223-33. doi: 10.1006/jmbi.1999.2706.
Ribosomal protein L11 is a highly conserved protein that has been implicated in binding of elongation factors to ribosomes and associated GTP hydrolysis. Here, we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S subunits by directed hydroxyl radical probing from Fe(II) tethered to five engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker 1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was analyzed by incorporating the modified proteins into 50 S subunits isolated from an E. coli strain that lacks L11 and testing for previously characterized L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA were detected by primer extension. Strong cleavages were detected within the previously described binding site of L11, in the 1100 region of 23 S rRNA. Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the sarcin loop in domain VI while probing from position 92 cleaved the backbone around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II. These data provide new information about the positions of specific features of 23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that L11 is near highly conserved elements of the rRNA that have been implicated in binding of tRNA and elongation factors to the ribosome.
核糖体蛋白L11是一种高度保守的蛋白质，它与延伸因子与核糖体的结合及相关的GTP水解有关。在此，我们通过从与1-(对溴乙酰氨基苄基)-EDTA连接子相连的五个工程化半胱氨酸残基（位于19、84、85、92和116位）处的Fe(II)进行定向羟基自由基探测，分析了大肠杆菌50 S亚基中L11的核糖体RNA邻域。通过将修饰后的蛋白质整合到从缺乏L11的大肠杆菌菌株中分离出的50 S亚基中，并测试23 S rRNA结构域II中先前表征的L11依赖性足迹，来分析L11衍生物的正确组装。由与L11相连的Fe(II)产生羟基自由基，并通过引物延伸检测核糖体RNA中的切割位点。在先前描述的L11结合位点内，即23 S rRNA的1100区域，检测到强烈的切割。此外，与L11中19位相连的Fe(II)靶向结构域VI中sarcin环的主干，而从92位进行探测时，分别切割了结构域II和V中900和2470位碱基周围的主干。与84、85和92位相连的Fe(II)也在螺旋II周围的5 S rRNA中产生了切割。这些数据提供了关于23 S rRNA和5 S rRNA特定特征相对于50 S亚基中蛋白质L11的位置的新信息，并表明L11靠近rRNA的高度保守元件，这些元件与tRNA和延伸因子与核糖体的结合有关。
