Quantifying the sequence-function relation in gene silencing by bacterial small RNAs
Quantifying the sequence-function relation in gene silencing by bacterial small RNAs
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DOI：
10.1073/pnas.1100432108
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发表时间：
2011-07-26
影响因子：
11.1
通讯作者：
Shi, Hualin
中科院分区：
文献类型：
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作者：
Hao, Yue;Zhang, Zhongge J.;Shi, Hualin
小RNA（sRNA）介导的基因沉默的序列-功能关系进行了量化的sRNA RyhB和它的一些mRNA的目标在大肠杆菌。产生了许多RyhB及其靶标的突变体，并且它们的体内功能以不同水平的靶标和RyhB表达为特征。虽然需要一个核心互补区的抑制RyhB，在核心区域的互补序列的变化引起了一个连续的抑制强度，与计算的自由能RyhB-目标双链体形成指数相关。此外，已知与RNA分子伴侣Hfq相互作用的接头区中的序列变异也引起了连续的抑制强度，与保持接头区开放的计算能量成本呈指数相关。这些结果支持了热力学模型在预测sRNA-mRNA相互作用中的适用性，并表明这些位置的序列可用于微调阻遏的程度。令人惊讶的是，发现没有Hfq结合区的截短的RyhB在存在和不存在Hfq的情况下都有效地抑制野生型RyhB的多个靶标，即使前者是野生型RyhB本身的活性所需的。这些发现挑战了普遍接受的模型，关于Hfq在基因沉默中的功能，既在提供稳定性的sRNAs，并在催化目标mRNA采取积极的构象，并提出了有趣的问题，为什么许多内源性sRNAs的主题，其功能的Hfq依赖。
Sequence-function relations for small RNA (sRNA)-mediated gene silencing were quantified for the sRNA RyhB and some of its mRNA targets in Escherichia coli. Numerous mutants of RyhB and its targets were generated and their in vivo functions characterized at various levels of target and RyhB expression. Although a core complementary region is required for repression by RyhB, variations in the complementary sequences of the core region gave rise to a continuum of repression strengths, correlated exponentially with the computed free energy of RyhB-target duplex formation. Moreover, sequence variations in the linker region known to interact with the RNA chaperone Hfq also gave rise to a continuum of repression strengths, correlated exponentially with the computed energy cost of keeping the linker region open. These results support the applicability of the thermodynamic model in predicting sRNA-mRNA interaction and suggest that sequences at these locations may be used to fine-tune the degree of repression. Surprisingly, a truncated RyhB without the Hfq-binding region is found to repress multiple targets of the wild-type RyhB effectively, both in the presence and absence of Hfq, even though the former is required for the activity of wild-type RyhB itself. These findings challenge the commonly accepted model concerning the function of Hfq in gene silencing-both in providing stability to the sRNAs and in catalyzing the target mRNAs to take on active conformations-and raise the intriguing question of why many endogenous sRNAs subject their functions to Hfq-dependences.