# Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress
## 3 Results
### Trx-h2 Melts to Nucleic Acids  
To test whether Trx-h2 can dissociate double-stranded nucleic acids, we used two partially complementary double-stranded oligonucleotides, which together comprised a molecular beacon. In the synthetic DNA substrate of the molecular beacon, the end of one strand was labeled with a fluorophore, while that of the other strand was conjugated with the fluorescent quencher (Figure S1). Since the fluorophore and quencher labeled at the end of the molecular beacon form a stem-loop structure, fluorescence is significantly extinguished by the quencher when the ends of the two strands form a stem-loop structure. However, melting of the annealed hairpin-like beacon by RNA chaperone allows the emission of the fluorophore because it is spatially separated from the quencher. To determine the nucleic acid melting activity of Trx-h2, we incubated the Trx-h2 protein with the molecular beacon (substrate) and measured the fluorescence emitted by the fluorophore. We also reacted the molecular beacon with GST and CspA as negative and positive controls, respectively. Both Trx-h2 and CspA remarkably increased the fluorescence intensity of the molecular beacon (Figure 5e). By contrast, Trx-h3 and GST did not increase the fluorescence intensity of the beacon, suggesting that Trx-h2 exhibits a strong dsDNA melting activity in vitro. Next, we examined the nucleic acid melting activity of Trx-h2 in vivo by using _E. coli_ RL211 cells containing the _chloramphenicol acetyltransferase_ (_CAT_) gene preceded by a \(\rho\)-independent _trpl_ terminator. At the _trpl_ termination sequences, two structural features of the transcribed RNA chain were detected. The transcribed RNA chain contained the GC-rich self-complementary area with several intruding nucleotides, which permitted to form a hairpin-like stem-loop structure. Immediately after the stem-loop secondary structure, several U bases were followed in the transcribed RNA. Then the hairpin-like structure interacted with the RNA polymerase and led to its stalling at the transcribing site. In particular, since the base pairs between the A bases in the template DNA and the 3'-U residues of the nascent RNA molecule are exceedingly precarious, they prompted the liberation of the RNA chain from the transcription complex at _trpl_ termination sites, without proceeding the _CAT_ gene transcription. The result makes the RL211 cells sensitive to chloramphenicol. However, when the RNA chaperone melted the hairpin-like stem-loop structure of the nascent RNA chain, it allows the continuous transcription of RNA polymerase for the _CAT_ gene. Then the bacteria can express the CAT protein and make the cells tolerant to chloramphenicol (Figure S2). For investigating the RNA-melting activity of Trx-h2, we cloned _Trx-h2_ into the _plNIII_ vector and transformed the DNA construct intoRL211 cells. Using the bacterial cells expressing the protein, we examined their survival in LB-agar media containing chloramphenicol, comparing with the cells expressing the _plNIII_ vector, _CspA_, and _Trx-h3_ as controls. Whereas the RL211 cells transforming the _plNIII_ vector or _Trx-h3_ construct were not able to grow on LB-agar medium containing chloramphenicol, the cells expressing _Trx-h2_ and _CspA_ grew well under the same conditions (Figure 5f). The results strongly suggested that Trx-h2 melted the hairpin-like secondary structure of _trpL_ terminator located at immediately upstream of the _CAT_ gene, which permitted the expression of CAT protein. Then, the bacterial cells expressing the CAT protein were able to grow at the presence of chloramphenicol. Taken together with the DNA-binding and melting activities of Trx-h2, our in vitro and in vivo data clearly show that the redox protein, Trx-h2 in Arabidopsis, functions as an RNA chaperone under cold conditions. Figure 5: Nucleic acid binding and melting activity of Trx-h2.