Structural organization and regulation of the plasmid-borne type II restriction-modification system Kpn21 from Klebsiella pneumoniae RFL2
Structural organization and regulation of the plasmid-borne type II restriction-modification system Kpn21 from Klebsiella pneumoniae RFL2
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DOI：
10.1093/nar/27.21.4228
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发表时间：
1999-11-01
影响因子：
14.9
通讯作者：
Janulaitis, A
中科院分区：
文献类型：
--
作者：
Lubys, A;Jurènaitè, S;Janulaitis, A
来自细菌肺炎克雷伯氏菌菌株RFL2的II型限制-修饰（R-M）系统的Kpn 21酶识别序列5 ′-TCCGGA-3 ′。克隆了Kpn 21 R-M基因并在大肠杆菌中表达，DNA序列分析表明，该基因含有两个可聚合转录的开放阅读框（ORF），编码301个氨基酸的限制性内切酶（Enase）（34.8 kDa）和375个氨基酸的甲基转移酶（MTase）（42.1 kDa），这些基因的3 '末端（分别为kpn21 R和kpn21 M）重叠11 bp。在kpn 21M上游发现一个小的ORF（基因kpn 21C），编码96个氨基酸（10.6kDa）的蛋白质。kpn 21C转录方向与kpn 21M相反。该ORF的预测氨基酸序列包括一个可能的螺旋-转角-螺旋基序。我们发现，kpn 21 C的产物抑制Kpn 21 MTase的表达，但对Enase基因的表达没有影响。这种调节方式在目前所分析的R-M系统中是独一无二的。肺炎杆菌RFL2质粒pKp4.3，其能够在大肠杆菌细胞中复制。
Kpn21 enzymes of a type II restriction-modification (R-M) system from the bacterium Klebsiella pneumoniae strain RFL2 recognize the sequence 5'-TCCGGA-3'. The Kpn21 R-M genes have been cloned and expressed in Escherichia coli, DNA sequence analysis revealed the presence of two convergently transcribed open reading frames (ORFs) coding for a restriction endonuclease (Enase) of 301 amino acids (34.8 kDa) and methyltransferase (Mtase) of 375 amino acids (42.1 kDa), The 3'-terminal ends of these genes (kpn21R and kpn21M, respectively) overlap by 11 bp, In addition, a small ORF (gene kpn21C) capable of coding for a protein of 96 amino acids in length (10.6 kDa) was found upstream of kpn21M, The direction of kpn21C transcription is opposite to that of kpn21M. The predicted amino acid sequence of this ORF includes a probable helix-turn-helix motif. We show that the product of kpn21C represses expression of the Kpn21 Mtase but has no influence on expression of the Enase gene. Such a mode of regulation is unique among R-M systems analyzed so far, The Kpn21 R-M is located on the K. pneumoniae RFL2 plasmid pKp4.3, which is able to replicate in E.coli cells.