Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Commissioned Corps, U.S. Public Health Service, Rockville, MD 20852, USA.
Nucleic Acids Res. 2019 Jan 10;47(1):310-327. doi: 10.1093/nar/gky1109.
RNA polymerase (RNAP) III synthesizes tRNAs and other transcripts, and mutations to its subunits cause human disorders. The RNAP III subunit-heterodimer C37/53 functions in initiation, elongation and in termination-associated reinitiation with subunit C11. C37/53 is related to heterodimers associated with RNAPs I and II, and C11 is related to TFIIS and Rpa12.2, the active site RNA 3' cleavage factors for RNAPs II and I. Critical to termination is stability of the RNA:DNA hybrid bound in the active center, which is loose for RNAP III relative to other RNAPs. Here, we examined RNAP III lacking C37/53/C11 and various reconstituted forms during termination. First, we established a minimal terminator as 5T and 3A on the non-template and template DNA strands, respectively. We demonstrate that C11 stimulates termination, and does so independently of its RNA cleavage activity. We found that C37/53 sensitizes RNAP III termination to RNA:DNA hybrid strength and promotes RNA 3' end pairing/annealing with the template. The latter counteracts C11-insensitive arrest in the proximal part of the oligo(T)-tract, promoting oligo(rU:dA) extension toward greater hybrid instability and RNA release. The data also indicate that RNA 3' end engagement with the active site is a determinant of termination. Broader implications are also discussed.
RNA 聚合酶 (RNAP) III 合成 tRNA 和其他转录物，其亚基的突变会导致人类疾病。RNAP III 亚基异二聚体 C37/53 参与起始、延伸以及与亚基 C11 相关的终止相关重新起始。C37/53 与与 RNAPs I 和 II 相关的异二聚体有关，而 C11 与 TFIIS 和 Rpa12.2 有关，它们是 RNAPs II 和 I 的活性位点 RNA 3' 切割因子。对于终止而言，关键是结合在活性中心的 RNA:DNA 杂交体的稳定性，相对于其他 RNAPs，RNAP III 的杂交体稳定性较弱。在这里，我们在终止过程中检查了缺乏 C37/53/C11 的 RNAP III 和各种重组形式。首先，我们在非模板和模板 DNA 链上分别建立了 5T 和 3A 作为最小终止子。我们证明 C11 可刺激终止，并且独立于其 RNA 切割活性。我们发现 C37/53 使 RNAP III 终止对 RNA:DNA 杂交体强度敏感，并促进 RNA 3' 末端与模板配对/退火。后者抵消了在寡聚（T）-链近端部分的 C11 不敏感停滞，促进寡聚（rU:dA）向更大的杂交体不稳定性和 RNA 释放的延伸。数据还表明，RNA 3' 末端与活性位点的结合是终止的决定因素。还讨论了更广泛的影响。
