DeLong A, Syvanen M
Department of Biology, Yale University, New Haven, CT 06511.
Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6072-6. doi: 10.1073/pnas.88.14.6072.
Transposition of Tn5 and of its component insertion sequence IS50R is regulated through the action of two proteins it encodes: a cis-acting transposase, Tnp, and a trans-acting inhibitor of transposition, Inh. The mechanism of the cis-acting Tnp and the relevance of inhibition to cis action have been addressed in the current study. A specific colony morphology assay for transposition of Tn5 was shown to be sensitive to Inh produced in trans and was used to screen for mutants in Inh and/or Tnp with altered regulation. A dominant mutant in IS50R that promotes transposition in trans was isolated and characterized. The mutant (449F) carries a Leu----Phe mutation at position 449 in Tnp. This mutation reduces the frequency of Tn5 or IS50R transposition in cis but allows Tnp-449F to act as efficiently in trans as it does in cis. Tnp-449F is sensitive to inhibition and, furthermore, Inh-449F is a competent inhibitor in trans. These results show that Tnp-449F is a trans-acting transposase, unlike wild-type Tnp, which is cis-acting.
Tn5及其组成部分插入序列IS50R的转座是通过它所编码的两种蛋白质的作用来调控的：一种顺式作用转座酶Tnp，以及一种转座的反式作用抑制剂Inh。本研究探讨了顺式作用Tnp的机制以及抑制作用与顺式作用的相关性。一种用于Tn5转座的特定菌落形态学检测方法被证明对反式产生的Inh敏感，并被用于筛选Inh和/或Tnp中调控改变的突变体。分离并鉴定了IS50R中一个促进反式转座的显性突变体。该突变体（449F）在Tnp的第449位携带一个Leu→Phe突变。此突变降低了Tn5或IS50R顺式转座的频率，但使Tnp - 449F在反式中的作用效率与在顺式中一样高。Tnp - 449F对抑制敏感，此外，Inh - 449F是一种有效的反式抑制剂。这些结果表明，与野生型Tnp（顺式作用）不同，Tnp - 449F是一种反式作用转座酶。
