Ganapathy Sweetha, Hoch Philipp G, Lechner Marcus, Bussiek Malte, Hartmann Roland K
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, 35037 Marburg, Germany.
Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, 35032 Marburg, Germany.
Noncoding RNA. 2022 Feb 16;8(1):20. doi: 10.3390/ncrna8010020.
Here we investigated the refolding of 6S-1 RNA and its release from σ-RNA polymerase (σ-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σ-RNAP affinity. This indicates that the RNA's extended helical arms including the '-35'-like region are not required for basic 6S-1 RNA functionality. The role of the 'central bubble collapse helix' (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σ-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5'-part of the CB (5'-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σ-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3'-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA's gel mobility and interfered with σ-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5'-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.
在此，我们使用截短和突变的6S-1 RNA变体，在体外研究了6S-1 RNA的重折叠及其从σ- RNA聚合酶（σ-RNAP）的释放。截短的6S-1 RNA仅由中央泡（CB）及其两侧的两个短螺旋臂组成，尽管其与σ-RNAP的亲和力降低了约10倍，但仍能在体外经历6S RNA的机制循环。这表明，6S-1 RNA的基本功能并不需要包括类似“-35”区域在内的RNA延伸螺旋臂。通过稳定突变研究了“中央泡塌陷螺旋”（CBCH）在pRNA诱导的6S-1 RNA从σ-RNAP重折叠和释放中的作用。这也揭示了在pRNA转录起始位点（第40位核苷酸）上游的CB（5'-CB）5'部分中的碱基身份，这些碱基身份会影响6S-1 RNA与σ-RNAP的基态结合。C44/45双突变对CBCH的稳定作用将pRNA长度模式转变为更短的pRNA，并且与弱化的P2螺旋相结合，导致从RNAP的释放更有效。我们得出结论，CBCH的形成支持pRNA诱导的6S-1 RNA重折叠和释放。我们的突变分析还揭示，在3'-CB中形成第二个短发夹对6S-1 RNA的释放不利。此外，当与6S-1 RNA退火时，短至6个核苷酸的pRNA的LNA模拟物会延迟RNA的凝胶迁移率并干扰σ-RNAP的结合。随着pLNA 7聚体和8聚体，这种作用逐渐增强，这表明通过与pRNA碱基配对引入到5'-CB中的构象灵活性受限，会阻止6S-1 RNA呈伸长形状。因此，对游离6S-1 RNA与6S-1：pLNA 8聚体和14聚体复合物的原子力显微镜观察表明，平均而言，6S-1：pRNA杂交结构比单独的6S-1 RNA具有更紧凑的结构。总体而言，我们的研究结果还表明，野生型6S-1 RNA序列和结构确保了6S-1 RNA机制循环中涉及的不同功能方面的最佳平衡。
