Modulation of the E. coli rpoH Temperature Sensor with Triptycene-Based Small Molecules

Abstract

Regulation of the heat shock response (HSR) is essential in all living systems. In E. coli, the HSR is regulated by an alternative σ factor, σ(32) , which is encoded by the rpoH gene. The mRNA of rpoH adopts a complex secondary structure that is critical for the proper translation of the σ(32) protein. At low temperatures, the rpoH gene transcript forms a highly structured mRNA containing several three-way junctions, including a rare perfectly paired three-way junction (3WJ). This complex secondary structure serves as a primitive but highly effective strategy for the thermal control of gene expression. In this work, the first small-molecule modulators of the E. coli σ(32) mRNA temperature sensor are reported.

Keywords: RNA; heat shock; temperature sensors; triptycenes.

Figure 1

(a) Brief schematic for the heat shock response in E. coli and strategy for small molecule modulation at the mRNA level. (b) Schematic showing the overall secondary structure of the 5′-end of the σ³² mRNA regulatory element. Important regions are shown with the boxed area corresponding to the AUG start codon.

Figure 2

Stabilization of a model system RNA by triptycene derivatives 1 and 2. (a) Structures of triptycene derivatives, Trip 1 and Trip 2. (b) RNA oligonucleotide used as a model system corresponding to a minimal sequence for junction formation. (c) UV thermal melting plots in the presence and absence of triptycenes. (d) Schematic of the fluorescence quenching experiment. (e) Titration of inhibitor 16 (I16) results in an increase in fluorescence. (f) Titration of Trip 1 or Trip 2 to the RNA*-I16 complex results in a decrease in fluorescence. The apparent K_d values of Trip 1 and Trip 2 were determined to be 2.5 μM and 1.5 μM, respectively.

Figure 3

Modulation of σ³² mRNA (-19 to +229) by triptycene derivatives and targeting σ³² in E. coli. (a) UV thermal melting plots in the absence and presence of Trip 1 or Trip 2. (b) Scheme for targeting rpoH using a σ³²-GFP fusion protein. (c) Relative fluorescence intensity of GFP control and σ³²-GFP fusion at 30 °C and 42 °C in the presence and absence of Trip 1 or Trip 2.