ermK leader peptide :: Amino acid sequence critical for induction by erythromycin
ermK leader peptide :: Amino acid sequence critical for induction by erythromycin
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DOI：
10.1007/bf02969307
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发表时间：
2006-12-01
影响因子：
6.7
通讯作者：
Choi, Eung-Chil
中科院分区：
文献类型：
--
作者：
Kwon, Ae-Ran;Min, Yu-Hong;Choi, Eung-Chil
来自地衣芽孢杆菌的ermK基因编码可诱导的rRNA甲基化酶，其赋予对大环内酯-林可酰胺-链阳性菌素B类抗生素的抗性。ermK mRNA前导序列的总长度为357个核苷酸，编码一个14个氨基酸的前导肽及其核糖体结合位点。ermK前导mRNA的二级结构和前导肽序列已被报道为控制表达的元件。在这项研究中，特定的前导肽氨基酸残基的诱导ermK的贡献进行了研究，使用基于PCR的大引物突变方法。将具有改变的前导肽密码子的ermK甲基化酶与大肠杆菌β-半乳糖苷酶报告基因互补融合。Thr-2 ~ Ser-4密码子的缺失降低了红霉素的诱导作用，而Thr-2和His-3密码子的缺失则无此作用。Ser-4、Met-5和Arg-6密码子的终止突变导致诱导作用的丧失，而Phe-9密码子的终止突变则恢复了红霉素的诱导作用。总的来说，这些发现表明残基4、5和6的密码子包含诱导的关键区域。Leu-7终止突变的ermK基因组成型表达。因此，核糖体停滞在密码子7似乎是重要的ermK诱导。
The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E coli beta-galactosidase reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not, The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.