Gu Z, Lovett P S
Department of Biological Sciences, University of Maryland, Catonsville 21228.
Mol Microbiol. 1992 Oct;6(19):2769-76. doi: 10.1111/j.1365-2958.1992.tb01456.x.
Chloramphenicol activates translation of cat-86 mRNA by stalling a ribosome in the leader of individual transcripts. Stalling triggers two sequential events: the destabilization of a region of secondary structure that sequesters the cat ribosome-binding site (RBS-C), and the initiation of cat translation. The site of drug-dependent ribosome stalling is dictated by the leader sequence, crb; crb causes a ribosome to stall with its aminoacyl site at leader codon 6. We demonstrate that induction requires the maintenance of a precise spatial relationship between crb and sequences within the left inverted repeat of the secondary structure. Therefore, destabilization of the secondary structure during chloramphenicol induction may result from the interaction of a stalled ribosome with a specific sequence in the secondary structure rather than from non-specific masking of RNA sequences. cat-86 regulation also depends on the distance that separates crb from RBS-C. This interval of 33 nucleotides was incrementally increased and decreased by mutations within a loop in the secondary structure. Shortening the distance between crb and RBS-C by three nucleotides reduced induction by half and a deletion of nine nucleotides abolished induction. Insertion mutations were without effect on induced expression but elevated basal expression. The results indicate that when the A site of a ribosome occupies leader codon 6 the secondary structure is destabilized and there is no interference with entry of a second ribosome at RBS-C. The data further demonstrate that when the A site of a ribosome in the leader is within 30 nucleotides of RBS-C, cat expression decreases. This decrease probably results from competition of the leader ribosome with the ribosome initiating cat translation. Our observations demonstrate that in wild-type cat-86 the distances between crb and the secondary structure, and between crb and RBS-C provide the precise spacing necessary to achieve three interdependent effects: the destabilization of the RNA secondary structure by a ribosome stalled at crb; a lack of competition between a ribosome stalled at crb and the initiating ribosome; and maintenance of a low, but measurable, basal level of cat expression. The spatial relationships identified as necessary for the regulation of cat-86 are conserved in the regulatory regions for five other inducible cat genes.
氯霉素通过使核糖体在单个转录本的前导区停滞来激活cat - 86 mRNA的翻译。停滞引发两个连续事件：隔离cat核糖体结合位点（RBS - C）的二级结构区域的去稳定化，以及cat翻译的起始。药物依赖性核糖体停滞的位点由前导序列crb决定；crb导致核糖体在其氨酰基位点位于前导密码子6处停滞。我们证明诱导需要维持crb与二级结构左反向重复序列内的序列之间精确的空间关系。因此，氯霉素诱导过程中二级结构的去稳定化可能是由于停滞的核糖体与二级结构中特定序列的相互作用，而不是由于RNA序列的非特异性掩盖。cat - 86的调控还取决于crb与RBS - C之间的距离。二级结构中一个环内的突变使这个33个核苷酸的间隔逐渐增加和减少。将crb与RBS - C之间的距离缩短三个核苷酸会使诱导降低一半，而缺失九个核苷酸则消除诱导。插入突变对诱导表达没有影响，但会提高基础表达。结果表明，当核糖体的A位点占据前导密码子6时，二级结构去稳定化，并且对第二个核糖体在RBS - C处的进入没有干扰。数据进一步证明，当前导区中核糖体的A位点距离RBS - C在30个核苷酸以内时，cat表达下降。这种下降可能是由于前导核糖体与起始cat翻译的核糖体竞争所致。我们的观察表明，在野生型cat - 86中，crb与二级结构之间以及crb与RBS - C之间的距离提供了实现三种相互依赖效应所需的精确间距：在crb处停滞的核糖体使RNA二级结构去稳定化；在crb处停滞的核糖体与起始核糖体之间缺乏竞争；以及维持低但可测量的cat基础表达水平。在其他五个可诱导cat基因的调控区域中，确定为cat - 86调控所必需的空间关系是保守的。
