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...
An experimental system was designed to measure in vivo termination efficiency (TE) of the Rho-independent terminator and position–function relations were quantified for the terminator tR2 in Escherichia coli. The terminator function was almost completely repressed when tR2 was located several base pairs downstream from the gene, and TE gradually increased to maximum values with the increasing distance between the gene and terminator. This TE–distance relation reflected a stochastic coupling of the ribosome and RNA polymerase (RNAP). Terminators located in the first 100 bp of the coding region can function efficiently. However, functional repression was observed when the terminator was located in the latter part of the coding region, and the degree of repression was determined by transcriptional and translational dynamics. These results may help to elucidate mechanisms of Rho-independent termination and reveal genomic locations of terminators and functions of the sequence that precedes terminators. These observations may have important applications in synthetic biology.
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