Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes
Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes
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DOI：
10.1093/nar/26.6.1421
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发表时间：
1998-03-15
影响因子：
14.9
通讯作者：
Bartholomew, B
中科院分区：
文献类型：
--
作者：
Tate, JJ;Persinger, J;Bartholomew, B
为了优化DNA光亲和标记检测蛋白质-DNA接触，我们将四种不同的光反应基团连接到DNA上，并检查它们交联酵母RNA聚合酶III（pol III）转录复合物的能力。将含有与5-氨基烯丙基脱氧尿苷偶联的芳基叠氮化物（AB-dUMP）、二苯甲酮（BP-dUMP）、全氟化芳基叠氮化物（FAB-dUMP）或二氮丙啶（DB-dUMP）的光反应性核苷酸掺入到SUP 4 tRNA（Tyr）基因bp-3/-2或+11处。与二氮丙啶的光交联揭示了聚合物III与DNA的接触，所述接触通过使用芳基叠氮化物、氟化芳基叠氮化物或二苯甲酮基团附接至DNA的DNA光亲和标记而未检测到。这些新的接触是pol III的82 kDa亚基与起始复合物中bp-3/-2处的DNA的接触，以及pol III的82、40（37）和31 kDa亚基与延伸复合物中bp +11处的DNA的接触，延伸复合物停滞在bp +17处。这些结果为pol III的82、34和31 kDa亚基的亚复合物位于活跃转录的Pol III的转录泡附近提供了证据，因为所有三种蛋白质在停滞的转录复合物的bp +11处交联。
In order to optimize the detection of protein-DNA contacts by DNA photoaffinity labeling, we attached four different photoreactive groups to DNA and examined their ability to crosslink yeast RNA polymerase III (pol III) transcription complexes. Photoreactive nucleotides containing an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB-dUMP) coupled to 5-aminoallyl deoxyuridine were incorporated into the SUP4 tRNA(Tyr) gene at bp -3/-2 or +11. Photo-crosslinking with diazirine revealed contacts of pol III with DNA that are not detected by DNA photoaffinity labeling using an aryl azide, fluorinated aryl azide or benzophenone group attached to DNA. These novel contacts were of the 82 kDa subunit of pol III with DNA at bp -3/-2 in the initiation complex and of the 82, 40(37) and 31 kDa subunits of pol III with DNA at bp +11 in elongation complexes stalled at bp +17. These results provide evidence for the subcomplex of the 82, 34 and 31 kDa subunits of pol III being positioned near the transcription bubble of actively transcribing Pol III, as all three proteins were crosslinked at bp +11 of the stalled transcription complex.