In vitro activity of the baculovirus late expression factor LEF-5
In vitro activity of the baculovirus late expression factor LEF-5
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DOI：
10.1128/jvi.76.24.12663-12675.2002
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发表时间：
2002-12-01
影响因子：
5.4
通讯作者：
Jin, JP
中科院分区：
文献类型：
--
作者：
Guarino, LA;Dong, W;Jin, JP
杆状病毒晚期表达因子LEF-5具有与真核转录延伸因子SII中的结构域同源的锌带。为了确定LEF-5是否是延伸因子，我们从细菌过表达系统中纯化了它，并将其加入纯化的杆状病毒RNA聚合酶中。LEF-5增加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是必不可少的。然而，与SII不同，LEF-5似乎不能使RNA聚合酶从固有的暂停位点逃逸。此外，LEF-5没有增加转录的小DNA结合配体，抑制在其他系统中的延伸或病毒DNA结合蛋白，抑制杆状病毒RNA聚合酶的存在下。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，但这并没有增加通过添加LEF-5。使用肝素防止再起始的起始测定和延伸测定表明，LEF-5仅在没有肝素的情况下才有活性。总之，这些结果表明，LEF-5的功能作为一个起始因子，而不是作为一个延伸因子。
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.