HIV-1 Nucleocapsid Protein NCp7 and Its RNA Stem Loop 3 Partner: Rotational Dynamics of Spin-Labeled RNA Stem Loop 3

Abstract

The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio <or=7, which is the threshold ratio for chaperone effects), the probe tumbling time markedly increased to several nanoseconds, signifying a NCP7/RNA complex with restricted motion even at the initially mobile 5' position. Increasing the ionic strength to shield the electrostatic attraction between polyanionic RNA and polycationic NCp7 eliminated this immobilization. Forming the immobilized >or=3:1 complex also required intact Zn fingers. Stopped-flow EPR kinetics with NCP7/RNA mixed at a 4:1 ratio showed the major phase of NCp7 interaction with RNA stem loop 3 occurred within 4 ms, a second phase occurred with a time constant of approximately 30 ms, and a slower immobilization, possibly concomitant with large complex formation, proceeded over seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for viral packaging and genome fabrication.

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

“…SDSL has matured as a technique for probing protein structure and dynamics (Fanucci and Cafiso 2006), and has also been applied to investigate nucleic acids (Sowa and Qin 2008). However, SDSL studies of protein/RNA complexes are limited (Edwards et al 2005;Xi et al 2008;Zhang et al 2008) Here, two nitroxide derivatives, R1a and R1b (Fig. 1C), were attached at sites within the N-and C-fragment of two N-peptides without disrupting the native complex.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…882 Conformational information for DNA and RNA structures has been obtained using oligonucleotides bearing a nitroxide spin label attached to C5 of dU, 883 and interactions between a stem loop of RNA and a zinc finger have been reported using a spin label attached via a terminal phosphorothioate. 884 Interactions between the restriction endonuclease EcoR1 and DNA has also been studied using EPR. 885 The rigid spin-label cytosine analogue (40) has been synthesised and forms stable base pairs with guanine, and was used to study the motion within an oligonucleotides that can adopt two different hairpin conformations.…”

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

“…NCp7 was prepared by solid phase peptide synthesis and with analysis methods described previously (29)(30)(31)(32). The final NCp7 concentration was determined by using an extinction coefficient of…”