In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis
In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis
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DOI：
10.4161/rna.8.5.16151
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发表时间：
2011-09-01
期刊：
影响因子：
4.1
通讯作者：
Hartmann, Roland K.
中科院分区：
文献类型：
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作者：
Beckmann, Benedikt M.;Burenina, Olga Y.;Hartmann, Roland K.
通过差异高通量RNA测序（dRNA-seq），我们已经鉴定了短至8-12个核苷酸的“产物RNA”（pRNA），其由枯草芽孢杆菌RNA聚合酶（RNAP）使用调节6S-1 RNA作为模板在体内合成。通过体外转录实验和北方印迹证实了dRNA-seq数据。在我们的文库中，我们无法检测到可能代表源自6S-2 RNA的pRNA的具有统计学意义的读数数量。而B能在体外以6S-2 RNA为模板合成pRNA。枯草杆菌（A）RNAP。6S-1 pRNA水平在指数期较低，在稳定期增加，在从稳定期长出期间爆发，表明pRNA合成是一种保守的调节机制，但比以前认为的更动态和微调的过程。大多数pRNA具有8-15 nt的长度，极少数长达24 nt。pRNA的平均长度从静止状态到生长状态有增加的趋势。pRNA的合成起始于6S-1 RNA的C40和6S-2 RNA的U41，分别产生具有5 '末端G或A残基的pRNA。A B。编码具有5 ′-末端A残基的pRNA的枯草杆菌6S-1 RNA突变株在不同生长状态之间显示出类似于14-nt pRNA的相同相对分布，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。C40 U突变体6S-1 RNA对sigma（A）RNAP的亲和力降低了两倍，这可能是pRNA水平降低的原因。我们推断，6S-1 pRNA的合成，虽然进化优化的起始与+1G残基，并不主要通过生长阶段依赖的变化在细胞GTP池的转录起始水平进行调节。
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 sigma(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 similar to 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 similar to two-fold lower affinity of the C40U mutant 6S-1 RNA towards sigma(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.