Mapping Col E1 RNA I - RNA II Kissing Complex and ROM Binding Interface using Paramagnetic Relaxation Enhancement NMR

Abstract

The RNA II transcript of ColE1 plasmid acts as the primer of plasmid replication, while RNA I transcript, an RNA antisense to the 5’ end of RNA II, acts as a suppressor of replication. The RNA I and RNA II interaction involves the formation of a kissing complex that is stabilized by plasmid encoded RNA One Modulator (ROM) protein. High-resolution structures of both the ROM protein and the RNA kissing complex have been determined. Alanine scanning mutagenesis experiments of ROM have shown that the surface residues Asn10, Phe14, Glu18, Lys25, and Lys3 of helix 1 interact with RNA loop residues. To date, there is no high resolution structure of the complex of ROM with the kissing complex and the details of this interaction remain unknown. Towards our effort to determine this RNA-protein complex, an RNA construct capable of forming a C2 symmetric kissing complex, to reduce NMR spectral complexity, has been designed by introducing a U-U mismatch into the kissing helix. 2D NMR spectra of the kissing dimer formed by this construct reveal a structural fold similar to the wild type. The challenge of elucidating a high resolution structure of this complex, however, lies in availability of few, if any, NOE contacts between ROM and RNA. To overcome this problem, the RNA UU kissing dimer construct has been 5′-end labeled with 3-(2-Iodoacetamido)-proxyl group so distance restraints could be measured using the paramagnetic relaxation enhancement (PRE) effect of the spin label on the protons in the protein. Using T1/T2 relaxation measurements, structural restraints for the complex are being pursued to enable a high resolution description of the how ROM specifically binds the RNA kissing complex.