Quantifying the sequence–function relation in gene silencing by bacterial small RNAs

Abstract

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 conformationsand raise the intriguing question of why many endogenous sRNAs subject their functions to Hfq-dependences.gene regulation | noncoding RNA | posttranscriptional control | quantitative biology | RNA interaction A significant development in gene regulation in the past decade is a growing appreciation of the complex roles that small regulatory RNA (sRNA) can play in coordinating gene activities in both prokaryotes and eukaryotes (1-3). In Escherichia coli, approximately 80 sRNA genes have been identified (3). There exists by now a basic understanding of the molecular components and mechanisms involved, at least for a major class of bacterial sRNA that acts in trans through base pairing (4-15). Recent theoretical and experimental studies have further revealed unique functional features of sRNA-mediated gene regulation (9,(16)(17)(18)(19)(20): because of the stoichiometric mode of target inactivation, sRNAmediated regulation exhibits an abrupt and sensitive response to input signals while being robust to stochastic fluctuations.How is this mode of regulation encoded in the molecular sequences of the sRNA and its targets? In the case of transcriptional regulation, a great deal is known quantitatively about the interaction between a DNA binding sequence (operator) and its cognate transcription factor (TF) and the regulatory consequences of this interaction: similarity of the operator to its "consensus sequence" determines its binding affinity to the cognate TF (21-24), and the latter in turn affects the rate of transcriptional initiation (25). Such knowledge, obtained b...

The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3′ poly(U) tail

“…We found that a 14-nt RNA oligo corresponding to the minimum base-pairing region of SgrS is able to inhibit ptsG translation without the help of Hfq in vitro (Maki et al 2010). More recently, it has been shown that a truncated RyhB in which internal portions other than the base-pairing region are largely deleted is able to repress efficiently the target sodB mRNA in the absence of Hfq in vivo (Hao et al 2011).…”

The ancestral SgrS RNA discriminates horizontally acquired Salmonella mRNAs through a single G-U wobble pair

“…The striking overlap with the previous ptsG data (38) lead us to conclude that the SgrS seed region, a critical RNA element that had already evolved in Salmonella, was acquired for the regulation of the horizontally acquired SopD virulence factor. Successful seed pairing of small RNAs with targets has been proposed to largely depend on the thermodynamic stability of the RNA duplex that is formed (41,42). Accordingly, most target search algorithms, including the popular RNAhybrid , which was established with 10 targets of RybB sRNA (44).…”

Efficient Search, Mapping, and Optimization of Multi-protein Genetic Systems in Diverse Bacteria

“…The apparent Boltzmann constant, b, has been measured as 0.45 AE 0.05 mol/kcal, which was confirmed in a second study (Hao et al, 2011). In practice, we use a proportional constant of 2,500 to generate a proportional scale where physiological common translation initiation rates vary between 1 and 100,000 au.…”