Gu Z, Harrod R, Rogers E J, Lovett P S
Department of Biological Sciences, University of Maryland--Baltimore County, Catonsville 21228.
J Bacteriol. 1994 Oct;176(20):6238-44. doi: 10.1128/jb.176.20.6238-6244.1994.
Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA.
可诱导的氯霉素抗性基因cat和cmlA受翻译衰减调控。对于这两个基因，必须被翻译以将核糖体传递至诱导位点的前导密码子指定了一种在体外抑制肽基转移酶的肽。这些肽的抗肽基转移酶活性被认为选择了对诱导至关重要的核糖体停滞位点。利用cat-86前导编码的5聚体肽MVKTD的变体，我们证明了体外抗肽基转移酶活性与同一肽在体内支持氯霉素诱导的能力之间的相关性。MVKTD与23S rRNA中的核苷酸2058、2059和2060结合。ermC甲基化酶对核苷酸2058的体内甲基化既不干扰cat-86的诱导，也不干扰肽对肽基转移酶的抑制。这种甲基化消除了体外通常在红霉素和MVKTD之间发生的竞争。MVKTD抑制几种真细菌、一种代表性古细菌物种以及真核生物酿酒酵母的肽基转移酶。嗜热脂肪芽孢杆菌支持cat-86的体内诱导，从这种革兰氏阳性嗜热菌的50S核糖体中酚抽提的RNA在肽基转移酶测定中具有催化活性且对肽抑制敏感。我们的结果表明，cat前导肽对肽基转移酶的抑制对诱导至关重要，并且这种活性可通过肽氨基酸序列的微小变化而改变。显示具有可被肽抑制的肽基转移酶的生物范围广泛，这表明该靶点是核糖体的高度保守组分，包括23S rRNA。
