Kontermann R E, Kobor M, Bautz E K
Institute of Molecular Genetics, University of Heidelberg, Germany.
Protein Sci. 1993 Feb;2(2):223-30. doi: 10.1002/pro.5560020211.
The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.
多亚基真核RNA聚合酶的最大亚基和第二大亚基参与与DNA模板及新生RNA链的相互作用。利用蛋白质免疫印迹法（Southwestern）DNA结合技术以及对细菌表达的融合蛋白进行硝酸纤维素滤膜结合分析，我们鉴定出果蝇RNA聚合酶II最大的215 kDa亚基上一个区域，该区域有可能非特异性结合核酸。这个核酸结合区域位于氨基酸残基309 - 384之间，在真核和细菌RNA聚合酶的最大亚基中高度保守。与大肠杆菌DNA聚合酶I参与结合新合成的DNA双链的DNA结合裂隙区域存在同源性，这间接证明了最大亚基的核酸结合区域在转录过程中参与与双链核酸的相互作用。该区域的非特异性DNA结合行为类似于在硝酸纤维素滤膜结合分析中观察到的天然酶的行为以及在蛋白质免疫印迹法中分离出的最大亚基的行为。高含量的碱性氨基酸残基与RNA聚合酶非特异性结合DNA的静电性质相符。
