Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.
Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.
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DOI：
10.1093/nar/gkv1182
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发表时间：
2016-01-29
影响因子：
14.9
通讯作者：
Erlacher MD
中科院分区：
文献类型：
--
作者：
Hoernes TP;Clementi N;Faserl K;Glasner H;Breuker K;Lindner H;Hüttenhofer A;Erlacher MD
RNA转录物中的核苷酸修饰在生命的所有三个领域的每一种生物中都存在。6-甲基胞嘧啶（m6 A）、5-甲基胞嘧啶（m5 C）和假尿苷（m6 A）是真核生物mRNA编码序列中高度丰富的核苷酸修饰，而m5 C和m6 A修饰也已在古细菌和细菌mRNA中发现。采用体外翻译实验，我们系统地研究了核苷酸修饰对翻译的影响。我们在细菌ErmCL mRNA的密码子内的三个位置中的每一个位置引入m5 C、m6 A、N端或2′-O-甲基化核苷酸，并分析它们对翻译的影响。根据相应的核苷酸修饰以及其在密码子内的位置，蛋白质合成保持不受影响或在修饰位点处过早终止，导致全长肽的量减少。在后一种情况下，核糖体复合物的足印分析与修饰密码子处的翻译停滞一致。当在一个密码子内引入多个核苷酸修饰时，观察到对翻译的附加抑制作用。我们还鉴定了m5 C修饰，当位于第二密码子位置时，其改变了相应密码子的氨基酸同一性。我们的研究结果表明，一种新的模式的基因调控的核苷酸修饰细菌的mRNA。
Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m6A), 5-methylcytosine (m5C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m5C and m6A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced m5C, m6A, Ψ or 2′-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m5C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs.