sRNA作为核糖开关调控纤维小体表达的分子机制

Non-coding RNA, especially riboswitch and sRNA, play an important regulatory role in the whole life of bacteria. Our recent discovery suggested that a free sRNA was transcribed in the 5'UTR of cip-cel gene cluster from cellulolytic clostridia, which encoded key cellulase components of a multienzyme complex called cellulosome. Our results indicated that this sRNA can potentially sense the availability of carbon sources to regulate transcription of its downstream cellulosomal genes as riboswitch. Although we found that there was a domain in the upstream of sRNA to control premature termination of transcription, this domain did not harbor any sequences of transcription terminator, which suggesting that the non-coding RNA takes sRNA-mediated way to control premature termination of transcription. Here we proposed to reveal the molecular mechanism and the genetic machineries that underpin this novel process. Firstly, we will address how the sRNA recognize the sugar molecules. Secondly, we will identify those elements controlling premature transcription termination. These aims will be accomplished by sreening of the sugar molecules recognized by sRNA, identifying RNA-sugar interaction sites and target-binding sites for premature thermination. The molecular mechanism of riboswitch-sRNA coupling regulation of cellulsome stoichiometry elucidated in ths project should deepen understanding of biological function of non-coding RNA, and lay the groundwork for the construction of cell factory to efficiently cellulose degradation.

非编码RNA，尤其是核糖开关和sRNA，在细菌的整个生命活动中发挥着重要的调控作用。项目申请人在厌氧纤维素降解梭菌中，发现编码纤维素酶复合体即纤维小体的关键基因簇cip-cel的5’UTR转录一个游离的sRNA，该sRNA潜在具有核糖开关的功能——能感受碳源利用的难易程度调控下游纤维小体基因的转录。同时在sRNA上游发现一个控制转录终止的结构域，但该结构域并未包含任何转录终止子序列，推测其可能采取基于sRNA的转录终止机制。为了阐明该非编码RNA耦合核糖开关和sRNA功能的作用机理，本项目拟围绕该sRNA如何识别糖信号分子和控制下游基因提前转录终止这两个核心问题，通过sRNA识别糖信号筛选、功能结构解析、作用靶点确定等，揭示sRNA作为核糖开关调控纤维小体表达的分子机制。该项目的实施将发现非编码RNA作为调控元件新的调控手段，并为高产纤维小体菌株的构建提供新的设计思路。

细菌非编码RNA，尤其是5’非翻译区（5’UTR）在转录和转录后水平参与调控细菌多个重要生理代谢途径。本项目以厌氧纤维素降解梭菌Ruminiclostridium cellulolyticum和Ruminiclostridium papyrosolvens为研究对象，解析了其两个纤维小体编码关键基因簇cip-cel及xyl-doc的5’UTR作为核糖开关调控下游各个基因转录的分子机制。根据项目申请书研究计划，我们在前期转录组数据发现cip-cel及xyl-doc的5’UTR转录游离sRNA的基础上，主要围绕cip-cel及xyl-doc的5’UTR的信号识别，RNA剪切机制和转录终止等方面进行研究。通过本项目的实施，首先在体外和体内分析cip-cel及xyl-doc的5’UTR在不同碳源下对下游基因的转录调控，确定其潜在识别的碳源信号。其次在5’UTR结构预测的基础上，对其进行了一系列的截短功能验证，精确定位了核糖核酸内切酶作用的核苷酸序列和潜在控制转录提前终止的结构区域。同时，在本项目实施过程中，共发表论文9篇，其中SCI论文7篇，并培养博士研究生1名，硕士研究生4名。通过本项目实施，初步揭示了5’UTR 转录的sRNA作为核糖开关调控纤维小体表达的分子机制，该研究不仅为将高产纤维小体菌株的构建提供新的设计思路和靶点，同时也丰富了非编码RNA作为调控元件在转录起始后调控基因转录的分子机制。