Guarino Linda A, Dong Wen, Jin Jianping
Departments of Biochemistry, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA.
J Virol. 2002 Dec;76(24):12663-75. doi: 10.1128/jvi.76.24.12663-12675.2002.
The baculovirus late expression factor LEF-5 has a zinc ribbon that is homologous to a domain in the eukaryotic transcription elongation factor SII. To determine whether LEF-5 is an elongation factor, we purified it from a bacterial overexpression system and added it to purified baculovirus RNA polymerase. LEF-5 increased transcription from both late and very late viral promoters. Two acidic residues within the zinc ribbon were essential for stimulation. Unlike SII, however, LEF-5 did not appear to enable RNA polymerase to escape from intrinsic pause sites. Furthermore, LEF-5 did not increase transcription in the presence of small DNA-binding ligands that inhibit elongation in other systems or viral DNA-binding proteins which inhibit the baculovirus RNA polymerase. Exonuclease activity assays revealed that baculovirus RNA polymerase has an intrinsic exonuclease activity, but this was not increased by the addition of LEF-5. Initiation assays and elongation assays using heparin to prevent reinitiation indicated that LEF-5 was active only in the absence of heparin. Taken together, these results suggest that LEF-5 functions as an initiation factor and not as an elongation factor.
杆状病毒晚期表达因子LEF-5具有一个锌带，该锌带与真核转录延伸因子SII中的一个结构域同源。为了确定LEF-5是否为延伸因子，我们从细菌过表达系统中纯化了它，并将其添加到纯化的杆状病毒RNA聚合酶中。LEF-5增加了晚期和极晚期病毒启动子的转录。锌带内的两个酸性残基对刺激至关重要。然而，与SII不同，LEF-5似乎不能使RNA聚合酶从内在的暂停位点逃逸。此外，在存在抑制其他系统延伸的小DNA结合配体或抑制杆状病毒RNA聚合酶的病毒DNA结合蛋白的情况下，LEF-5不会增加转录。核酸外切酶活性测定表明杆状病毒RNA聚合酶具有内在的核酸外切酶活性，但添加LEF-5不会增加这种活性。使用肝素防止重新起始的起始测定和延伸测定表明，LEF-5仅在不存在肝素的情况下具有活性。综上所述，这些结果表明LEF-5作为起始因子发挥作用，而不是作为延伸因子。
