原核表达调控与色氨酸操纵子.ppt

弱化作用 trp操纵子转录终止的调控是通过弱化作用（attenuation）实现的。枯草杆菌色氨酸操纵子表达主要受到色氨酸激活RNA结合蛋白(Trp -activated RNA - binding p rotein,TRAP）的调节。该蛋白与色氨酸结合被激活后，可与trpE上游转录产物结合，导致转录终止。当色氨酸浓度较低时，TRAP失活，转录可以继续，结构基因得以表达。 反馈抑制作用 由于基因表达必然消耗一定的能源和前体物，相对于阻遏和弱化作用，反馈抑制作用更为经济和高效。终产物Trp对催化分支途径几步反应的酶具有反馈抑制作用，其50%抑制浓度分别为：邻氨基苯甲酸合酶，0. 0015 mmol·L - 1 ；邻氨基苯甲酸磷酸核糖转移酶，0. 15 mmol·L - 1 ;色氨酸合成酶,7. 7mmol·L - 1。对于普通野生菌株，邻氨基苯甲酸合酶对Trp合成起到关键调控作用，常被称为瓶颈酶；但对高产Trp工程菌而言，上述任何一种酶的反馈抑制都会直接影响Trp产量。 The Bacillus subtilis TRAP Protein Can Induce Transcription Termination in the Leader Region of the Tryptophan Biosynthetic (trp) Operon Independent of the trp Attenuator RNA 2014 1. In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism. 2. When intracellular tryptophan levels are high, the TRAP( trp RNA-binding attenuation protein ) protein binds to the 5‘ leader region of the nascent trp mRNA and induces transcription termination. 3. In limiting tryptophan, TRAP does not bind and the operon is transcribed. Two competing RNA secondary structures termed the antiterminator and terminator (attenuator) can form in the leader region RNA. Research Background 4. In prior attenuation models, the only role of TRAP binding was to alter the RNA secondary structure to allow formation of the attenuator, which has been thought function as an intrinsic transcription terminator. 5. However,recent studies have shown that the attenuator is not an effective intrinsic terminator. 6.From these studies it was not clear whether TRAP functions independently or requires the presence of the attenuator RNA structure. Figure 1. Model of transcription attenuation of the B. subtilis trp operon. Research Purposes 1. To examine the role of the attenuator RNA in TRAP-mediated transcription termination, to examine whether TRAP functions independently or requires the presence of the attenuator RNA structure. Material and Methods 1.Bacterial strains and transformations ?E. coli K802 was u