Pseudouridylation of helix 69 of 23S rRNA is necessary for an effective translation termination
Pseudouridylation of helix 69 of 23S rRNA is necessary for an effective translation termination
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DOI：
10.1073/pnas.0706558104
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发表时间：
2007-12-04
影响因子：
11.1
通讯作者：
Pedersen, Steen
中科院分区：
文献类型：
--
作者：
Ejby, Morten;Sorensen, Michael A.;Pedersen, Steen
先前发现，rud基因失活的大肠杆菌菌株缺乏23S核糖体RNA螺旋69上保守的假尿嘧啶，生长缓慢。一个生长速度接近正常的抑制突变体将prfB中保守的Glu-172密码子改变为编码翻译终止因子RF2的Lys密码子。当分析prfB(+)/prfB（E172K）和rluD(+)/rluD::cat组合菌株的无义抑制时，发现rluD失活增加了所有三个终止密码子作为意义密码子的误读：UAG密码子的无义抑制增加了2倍，UAA增加了9倍，UGA增加了14倍。在30%的情况下，增加的UGA通读对应于将UGA读取为sense密码子。相反，读义密码子的准确性没有受到rluD缺失的影响。当失活的rluD基因与改变的prfB结合时，在UAA密码子处恢复了野生型的终止水平，并且在UGA处的终止效率高于野生型。这些结果有力地表明，至少有一个螺旋69伪尿嘧啶在翻译终止中起作用。据我们所知，这是第一次描述核糖体RNA伪尿嘧啶修饰的功能。
Escherichia coli strains with inactivated rluD genes were previously found to lack the conserved pseudouridines in helix 69 of 23S ribosomal RNA and to grow slowly. A suppressor mutant was isolated with a near normal growth rate that had changed the conserved Glu-172 codon to a Lys codon in prfB, encoding translation termination factor RF2. When nonsense suppression in strains with all combinations of prfB(+)/prfB(E172K) and rluD(+)/rluD::cat was analyzed, misreading of all three stop codons as sense codons was found to be increased by rluD inactivation: Nonsense suppression was increased 2-fold at UAG codons, 9-fold at UAA, and 14-fold at UGA. The increased read-through at UGA corresponds to reading UGA as a sense codon in 30% of the cases. In contrast, the accuracy of reading sense codons appeared unaffected by loss of rluD. When the inactivated rluD gene was combined with the altered prfB, wild-type levels of termination were restored at UAA codons and termination was more efficient than wild type at UGA. These results strongly suggest that at least one of the helix 69 pseudouridines has a function in translation termination. To our knowledge, this is the first described function for a ribosomal RNA pseudouridine modification.