Alexieva Z, Duvall E J, Ambulos N P, Kim U J, Lovett P S
Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
Proc Natl Acad Sci U S A. 1988 May;85(9):3057-61. doi: 10.1073/pnas.85.9.3057.
The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. Induction by the antibiotic is primarily due to activation of the translation of cat-86-encoded mRNA. It has been suggested that the inducer stalls ribosomes at a discrete location in the leader region of cat-86 mRNA, which causes the destabilization of a downstream RNA secondary structure that normally sequesters the cat-86 ribosome binding site. It is the destabilization of this RNA secondary structure that permits translation of the cat-86 coding sequence. In the present report, we show that ribosomes that were stalled in the cat-86 leader by starvation of host cells for the amino acid specified by leader codon 6 induced gene expression to a level above that detected when cells were starved for the amino acids specified by leader codons 7 and 8. Starvation for amino acids specified by leader codons 3, 4, or 5 failed to activate cat-86 expression. These results indicate that the stalled ribosome that is most active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To determine if chloramphenicol also stalled ribosomes in the cat-86 regulatory leader such that the aminoacyl site was occupied by codon 6, we separately changed leader codons 3, 4, 5, and 6 to the translation termination (ochre) codon TAA. Each of the mutated genes was tested for its ability to be induced by chloramphenicol. The results show that replacement of leader codons 3, 4, or 5 by the ochre codon blocked induction, whereas replacement of leader codon 6 by the ochre codon permitted induction. Collectively, these observations lead to the conclusion that cat-86 induction requires ribosome stalling in leader mRNA, and they identify leader codon 6 as the codon most likely to be occupied by the aminoacyl site of a stalled ribosome that is active in the induction.
质粒基因cat - 86在枯草芽孢杆菌中编码氯霉素诱导型氯霉素乙酰转移酶。抗生素诱导主要是由于cat - 86编码的mRNA翻译激活。有人提出，诱导剂使核糖体在cat - 86 mRNA前导区的一个离散位置停滞，这导致下游RNA二级结构不稳定，该二级结构通常会隔离cat - 86核糖体结合位点。正是这种RNA二级结构的不稳定使得cat - 86编码序列能够翻译。在本报告中，我们表明，由于宿主细胞缺乏前导密码子6所指定的氨基酸而在cat - 86前导区停滞的核糖体诱导基因表达的水平高于细胞缺乏前导密码子7和8所指定的氨基酸时检测到的水平。缺乏前导密码子3、4或5所指定的氨基酸未能激活cat - 86表达。这些结果表明，在cat - 86诱导中最活跃的停滞核糖体的氨酰基位点被前导密码子6占据。为了确定氯霉素是否也使核糖体在cat - 86调控前导区停滞，使得氨酰基位点被密码子6占据，我们分别将前导密码子3、4、5和6替换为翻译终止（赭石）密码子TAA。测试了每个突变基因被氯霉素诱导 的能力。结果表明，用赭石密码子替换前导密码子3、4或5会阻断诱导，而用赭石密码子替换前导密码子6则允许诱导。总的来说，这些观察结果得出结论，cat - 86诱导需要核糖体在前导mRNA中停滞，并且它们确定前导密码子6是最有可能被在诱导中活跃的停滞核糖体的氨酰基位点占据的密码子。
