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首页> 外文期刊>Biochemistry >EVALUATION HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE TRANSCRIPTASE PRIMER TRNA BINDING BY FLUORESCENCE SPECTROSCOPY - SPECIFICITY AND COMPARISON TO PRIMER/TEMPLATE BINDING
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EVALUATION HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE TRANSCRIPTASE PRIMER TRNA BINDING BY FLUORESCENCE SPECTROSCOPY - SPECIFICITY AND COMPARISON TO PRIMER/TEMPLATE BINDING

机译：荧光光谱法评价人类免疫缺陷病毒1型逆转录酶原核糖核酸的结合-特异性及其与模板结合的比较。

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A host cell-derived tRNA(3)(Lys) molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome, We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA(3)(Lys), and to address RT's putative role in the required and specific packaging of tRNA(3)(Lys) into the budding virus, Titration of RT with tRNA(3)(Lys) resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (K-d) Of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA(2)(Glu), E. coli tRNA(2)(Tyr), E. coli tRNA(Lys), yeast tRNA(Phe), Or in vitro-synthesized human tRNA(3)(Lys) (no base modifications) resulted in similar fluorescence changes and K-d values as obtained for the natural tRNA(3)(Lys). The specific interaction between RT and tRNA(3)(Lys) during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA(3)(Lys). The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNA(Phe) was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites, Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA and p/t binding. Reverse transcriptase was found to bind to the mutant tRNA 10-fold more tightly than to the unaltered tRNA (K-d = 4.5 +/- 1.0 and 44.6 +/- 6.6 nM, respectively). Further analyses revealed that the tighter affinity is probably due to a preferred p/t binding mode and not to one expected if separate tRNA and p/t binding regions are accessed simultaneously by the same molecule.
机译：人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）利用源自宿主细胞的tRNA（3）（Lys）分子引发病毒RNA基因组的DNA合成，我们进行了荧光滴定实验以表征相互作用在RT及其天然引物tRNA（3）（Lys）之间进行定位，以解决RT在将tRNA（3）（Lys）所需和特定包装入萌芽病毒中的推定作用，用tRNA（3）（Lys滴定）RT ）导致RT色氨酸荧光最大猝灭30％，由此可得出57.6 +/- 7.5 nM的解离常数（Kd）。用大肠杆菌tRNA（2）（Glu），大肠杆菌tRNA（2）（Tyr），大肠杆菌tRNA（Lys），酵母tRNA（Phe）或体外合成的人tRNA（3）滴定RT Lys）（无碱基修饰）产生与天然tRNA（3）（Lys）类似的荧光变化和Kd值。因此，RT和tRNA（3）（Lys）之间在病毒组装过程中先前的体内研究表明的特异性相互作用在RT的完全加工体外形式中不存在。因此，病毒组装过程中的其他因素必须在tRNA（3）（Lys）的包装中发挥作用。在本研究中检测到的非特异性和离子强度依赖性的RT-tRNA相互作用表明，tRNA的整体形状和电荷构成RT结合的识别特征。发现RT结合后，酵母tRNA（Phe）的反密码子环上所含的维特丁碱的荧光增强，从而推测RT与tRNA的反密码子环相互作用。单个tRNA还从RT移出了荧光DNA引物/模板（p / t）底物，表明tRNA和p / t结合位点重叠。置换滴定的立方拟合评估可以进一步评估两个竞争配体的亲和力。通过检查可能的RT双底物类型抑制剂的亲和力来测试RT上重叠的p / t和tRNA结合区以及分开的p / t和tRNA结合区的存在，该抑制剂包含对tRNA和p / t结合必不可少的基序。发现逆转录酶与突变tRNA的结合比未改变tRNA的结合更紧密10倍（K-d分别为4.5 +/- 1.0和44.6 +/- 6.6 nM）。进一步的分析表明，更紧密的亲和力可能是由于一种优选的p / t结合模式所致，而不是由于同一分子同时访问分开的tRNA和p / t结合区而导致的。

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