Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2011 May 6;286(18):16109-20. doi: 10.1074/jbc.M111.228023. Epub 2011 Mar 18.
Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1-270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1-380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.
酵母（酿酒酵母）线粒体（mt）基因组的转录由核编码蛋白催化，这些蛋白包括核心 RNA 聚合酶（RNAP）亚基 Rpo41 和转录因子 Mtf1。Rpo41 与单亚基噬菌体 T7/T3 RNAP 同源。其约 80kDa 的 C 端结构域在 mt RNAP 中高度保守，但约 50kDa 的 N 端结构域（NTD）则不太保守，并且在 T7/T3 RNAP 中不存在。为了了解 NTD 的作用，我们对 Rpo41 的一系列 NTD 缺失突变体进行了生化表征。我们的研究表明，NTD 调节转录起始的多个步骤。有趣的是，NTD 在起始过程中以自动抑制的方式发挥作用，其部分缺失会提高 RNA 合成的效率。缺失 1-270 个氨基酸（DN270）相对于全长（FL）Rpo41 减少了无效合成，并增加了全长到无效 RNA 的比值。更大的缺失 1-380 个氨基酸（DN380）会降低双链体上的 RNA 合成，但不会降低预熔启动子上的 RNA 合成。我们表明，DN380 在转录起始位点附近的启动子打开中存在缺陷。最引人注目的是，DN270 和 DN380 都在预熔启动子上催化高度连续的 RNA 合成，与 FL Rpo41 不同，突变体不受 Mtf1 的抑制。两种突变体与 Mtf1 的相互作用较弱，这解释了我们的许多结果，特别是突变体有效地从起始过渡到延伸的能力。我们提出，在体内，与 NTD 结合的辅助蛋白可能会调节 Rpo41 与启动子/Mtf1 的相互作用，以激活并允许及时从 Mtf1 释放以过渡到延伸。
