AFFINITIES OF TRANSFER-RNA BINDING-SITES OF RIBOSOMES FROM ESCHERICHIA-COLI
AFFINITIES OF TRANSFER-RNA BINDING-SITES OF RIBOSOMES FROM ESCHERICHIA-COLI
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DOI：
10.1021/bi00359a025
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发表时间：
1986-06-03
期刊：
影响因子：
2.9
通讯作者：
WINTERMEYER, W
中科院分区：
文献类型：
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作者：
LILL, R;ROBERTSON, JM;WINTERMEYER, W
在不同的离子条件下，测定了来自大肠杆菌或酵母的tRNAPhe-tRNAPhe和N-AcPhe-tRNAPhe与E.Coli70S核糖体的P、A和E位的结合亲和力。对于滴定，使用了平衡(荧光)和非平衡(过滤)技术。通过利用三个结合位点的不同亲和力、稳定性和特异性来确定特定结合常数而不是化学计量结合常数。多聚(U)编程性核糖体的P位与tRNAPhe和N-AcPhe-tRNAPhe结合，结合常数分别为108M-1和5倍。109M-1。根据镁离子浓度的不同，与A位点的结合弱10-200倍。Phe-tRNAPhe以类似的亲和力结合到A位点。通过添加延伸因子Tu和GTP，将Phe-tRNAPhe的A位结合与GTP水解偶联，使平衡常数明显增加至少104倍。当省略聚(U)时，根据离子条件，P位的亲和力降低2-4个数量级，而A位的结合不再被检测到。与脱酰化的tRNAPhe特异结合的E位点的亲和力与A位点相当。与P位和A位相比，与E位的结合是不稳定的，对离子强度的变化不敏感。缺失该基因最多使亲和力降低4倍，表明E位不存在有效的密码子-反密码子相互作用。根据平衡常数，易位的位移步骤需要离开P位的tRNA与E位结合。在活体条件下，离开的tRNA与E位点或相关位点的瞬时结合的功能作用最有可能是提高易位的速度。
The binding affinities of tRNAPhe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 108 M-1 and 5 .times. 109 M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 104. Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficienct codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.