Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany.
RNA Biol. 2011 Sep-Oct;8(5):839-49. doi: 10.4161/rna.8.5.16151. Epub 2011 Sep 1.
By differential high-throughput RNA sequencing (dRNA-seq) we have identified "product RNAs" (pRNAs) as short as 8-12 nucleotides that are synthesized by Bacillus subtilis RNA polymerase (RNAP) in vivo using the regulatory 6S-1 RNA as template. The dRNA-seq data were confirmed by in vitro transcription experiments and Northern blotting. In our libraries, we were unable to detect statistically meaningful numbers of reads potentially representing pRNAs derived from 6S-2 RNA. However, pRNAs could be synthesized in vitro from 6S-2 RNA as template by the B. subtilis σ(A) RNAP. 6S-1 pRNA levels are low during exponential, increase in stationary, and burst during outgrowth from stationary phase, demonstrating that pRNA synthesis is a conserved regulatory mechanism, but a more dynamic and fine-tuning process than previously thought. Most pRNAs have a length of 8-15 nt, very few up to 24 nt. The average length of pRNAs tended to increase from stationary to outgrowth conditions. Synthesis of pRNA is initiated at C40 of 6S-1 RNA and U41 of 6S-2 RNA, yielding pRNAs with a 5'-terminal G or A residue, respectively. A B. subtilis 6S-1 RNA mutant strain encoding a pRNA with a 5'-terminal A residue showed the same relative distribution of ~14-nt pRNAs between the different growth states, but generally displayed lower pRNA levels than the reference strain encoding wild-type 6S-1 RNA. A ~two-fold lower affinity of the C40U mutant 6S-1 RNA towards σ(A) RNAP may have contributed to this reduction in pRNA levels. We infer that 6S-1 pRNA synthesis, although evolutionarily optimized for initiation with a +1G residue, is not primarily regulated at the transcription initiation level via growth phase-dependent variations in the cellular GTP pool.
通过差异高通量 RNA 测序（dRNA-seq），我们鉴定了“产物 RNA”（pRNA），其长度最短为 8-12 个核苷酸，在体内由枯草芽孢杆菌 RNA 聚合酶（RNAP）以调控 6S-1 RNA 为模板合成。dRNA-seq 数据通过体外转录实验和 Northern blot 得到验证。在我们的文库中，我们无法检测到来自 6S-2 RNA 的潜在有意义数量的读取，这些读取可能代表 pRNA。然而，pRNA 可以作为模板，由枯草芽孢杆菌 σ(A) RNAP 在体外从 6S-2 RNA 中合成。在指数生长期，pRNA 的水平较低，在静止期增加，并在从静止期到生长的过程中爆发，这表明 pRNA 合成是一种保守的调控机制，但比以前认为的更为动态和精细的过程。大多数 pRNA 的长度为 8-15 nt，极少数达到 24 nt。pRNA 的平均长度从静止期到生长条件下趋于增加。pRNA 的合成起始于 6S-1 RNA 的 C40 和 6S-2 RNA 的 U41，分别产生 5'-末端 G 或 A 残基的 pRNA。枯草芽孢杆菌 6S-1 RNA 突变株编码 5'-末端 A 残基的 pRNA，在不同生长状态下产生的~14-nt pRNA 的相对分布相同，但通常显示出比编码野生型 6S-1 RNA 的参考菌株更低的 pRNA 水平。C40U 突变 6S-1 RNA 对 σ(A) RNAP 的亲和力降低约两倍，可能导致 pRNA 水平降低。我们推断，6S-1 pRNA 合成虽然在进化上已优化为以+1G 残基起始，但主要不是通过细胞 GTP 池在生长阶段的变化来调节转录起始水平。
