Nanomechanics of the MPMV Capsid-Protein-Binding RNA Fragment

Abstract

The interaction between RNA and binding proteins plays an important role in the structure, stability, function and dynamics of either molecular species. The Mason-Pfizer monkey retrovirus (MPMV) is an ideal model for investigating RNA-protein interactions, because part of the genomic RNA, called the packaging signal, is influenced by the binding of its capsid proteins. To explore the properties of MPMV RNA, we manipulated individual molecules of the packaging-signal sequence by using force-measuring optical tweezers.The 207-base-long segment of MPMV RNA corresponding to the packaging signal, extended on each side with 1200-base-long indifferent gene segments for mechanical handling, was cloned into a pET28a vector, then expressed in an in vitro transcription system. Complementary DNA strands were hybridized to this RNA in a thermal-ramp protocol so as to obtain RNA/DNA handle ends. The complex was then manipulated in repetitive stretch and relaxation cycles across a force range of 0-80 pN under Mg-free conditions. At a stretch rate of 250 nm/s force increased monotonically and non-linearly. Above 15 pN a two-step transition was typically observed, corresponding to the unfolding of the RNA molecule. The total length gain (∼100 nm) associated with the process is in good agreement with the theoretical contour length. The transitions were not reversible during relaxation with a rate of 250 nm/s, suggesting that the force-dependent refolding kinetics are slow in comparison with that of the experiment. However, upon introducing a ∼30-second pause before the second mechanical cycle, the unfolding transitions reappeared. Our findings suggest that the packaging signal forms a complex, stable secondary structure that unfolds in at least two major steps. Whether the kinetics of the transitions are influenced by the presence of capsid proteins is a subject of further detailed investigations.