Wang Yanping, Shoemaker Nadja B, Salyers Abigail A
Department of Microbiology, B103 CLSL, University of Illinois, 601 S. Goodwin Avenue, Urbana, IL 61801, USA.
J Bacteriol. 2004 May;186(9):2548-57. doi: 10.1128/JB.186.9.2548-2557.2004.
CTnDOT is a conjugative transposon (CTn) that is found in many Bacteroides strains. Transfer of CTnDOT is stimulated 100- to 1,000-fold if the cells are first exposed to tetracycline (TET). Both excision and transfer of CTnDOT are stimulated by TET. An operon that contains a TET resistance gene, tetQ, and two regulatory genes, rteA and rteB, is essential for control of excision and transfer functions. At first, it appeared that RteA and RteB, which are members of a two-component regulatory system, might be directly responsible for the TET effect. We show here, however, that neither RteA nor RteB affected expression of the operon. TetQ, a ribosome protection type of TET resistance protein, actually reduced operon expression, possibly by interacting with ribosomes that are translating the tetQ message. Fusions of tetQ with a reporter gene, uidA, were only expressed at a high level when the fusion was cloned in frame with the first six codons of tetQ. However, out of frame fusions or fusions ending at the other five codons of tetQ showed much lower expression of the uidA gene. Moreover, reverse transcription-PCR amplification of tetQ mRNA revealed that despite the fact that the uidA gene product, beta-glucuronidase (GUS), was produced only when the cells were exposed to TET, tetQ mRNA was produced in both the presence and absence of TET. Computer analysis of the region upstream of the tetQ start codon predicted that the mRNA in this region could form a complex RNA hairpin structure that would prevent access of ribosomes to the ribosome binding site. Mutations that abolished base pairing in the stem that formed the base of this putative hairpin structure made GUS production as high in the absence of TET as in TET-stimulated cells. Compensatory mutations that restored the hairpin structure led to a return of regulated production of GUS. Thus, the tetQ-rteA-rteB operon appears to be regulated by a translational attenuation mechanism.
CTnDOT是一种接合转座子（CTn），存在于许多拟杆菌菌株中。如果细胞先暴露于四环素（TET），CTnDOT的转移会被刺激100到1000倍。CTnDOT的切除和转移都受到TET的刺激。一个包含TET抗性基因tetQ以及两个调控基因rteA和rteB的操纵子对于控制切除和转移功能至关重要。起初，似乎作为双组分调控系统成员的RteA和RteB可能直接负责TET效应。然而，我们在此表明，RteA和RteB都不影响该操纵子的表达。TetQ是一种核糖体保护型的TET抗性蛋白，实际上可能通过与翻译tetQ信息的核糖体相互作用而降低操纵子的表达。tetQ与报告基因uidA的融合体只有在与tetQ的前六个密码子读框内克隆时才会高水平表达。然而，读框外融合或在tetQ的其他五个密码子处终止的融合体显示uidA基因的表达要低得多。此外，tetQ mRNA的逆转录 - PCR扩增显示，尽管只有当细胞暴露于TET时才产生uidA基因产物β - 葡萄糖醛酸酶（GUS），但tetQ mRNA在有TET和无TET的情况下都会产生。对tetQ起始密码子上游区域的计算机分析预测，该区域的mRNA可形成一个复杂的RNA发夹结构，这会阻止核糖体接近核糖体结合位点。消除构成该假定发夹结构底部茎环中碱基配对的突变使得在无TET时GUS的产生与在TET刺激的细胞中一样高。恢复发夹结构的补偿性突变导致GUS的调控产生恢复。因此，tetQ - rteA - rteB操纵子似乎受翻译衰减机制调控。
