RNA-protein recognition/virus assembly

Hugo Lago, Tim Moss, Andrew M. Parrott, Ben Wittaker, Jenny Baker, Andy Baron,
Alison Ashcroft, Peter Stockley

 

The MS2 translational repression complex, that also serves as an assembly initiation complex, continues to provide unique insights into the details of sequence-specific RNA-protein recognition. In the past year, we have shown that a series of RNA aptamers, that mimic the natural operator binding but have distinct tertiary structures, have similar solution behaviour, as judged by the fluorescence emission spectra of RNAs modified with 2-aminopurine. That is, aptamers seem to be mimicking solution dynamic behaviour even when not liganded by the coat protein.

 

Ribbon diagram of a coat protein dimer bound to operator RNA. The RNA can be seen as a crescent, bound across the protein dimer.

Fluorescence spectroscopy, this time using stopped-flow kinetics, has also been used to probe the early events occurring when a coat protein dimer interacts with the RNA operator. Unexpectedly, it appears that a fraction of the coat protein molecules are unable to bind RNA even though the interaction is essentially diffusion driven, until they have undergone a conformational change. This is the first evidence linking RNA-binding with protein conformation and may provide insights into the mechanism of phage capsid self-assembly. To probe these secondary events we have been using Q-tof mass spectrometry to characterise higher order protein-RNA complexes. These studies are still on–going but it is clear that the Q-tof will be a major tool in analysing such assembly events.

Structure determination of an RNA chemically variant complex, in association with our longstanding collaborators in Sweden, provides strong evidence that understanding this important class of interactions will require extensive knowledge of both liganded and unliganded structures, since removal of a single hydrogen bonding group from a single base in the RNA results in major rearrangement of the RNA-protein interface. Electrostatic potential calculations of the effect of the chemical change suggest that the result is driven by the generation of unfavourable contacts in the unliganded RNA, and implies that caution will be needed in future in interpreting the results of such chemical variation experiments.

Collaborators

Nicola Stonehouse
Simon Phillips
Stuart Warriner
David Peabody (New Mexico)
Andy Ellington (Texas)
Darrell Davis (Arizona)
Leo Beigelmann (Colorado)
Lars Liljas (Uppsala)

Publications

Parrott, A.M., Lago, H., Adams, C.J., Ashcroft, A.E., Stonehouse, N.J. and Stockley, P.G. (2000). "RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour." Nuc. Acids Res. 28, 489-497.

Lago, H., Parrott, A.M., Moss, T., Stonehouse, N.J. and Stockley, P.G. (2001). "Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: Identification of a protein conformer unable to bind RNA." J.Mol.Biol 305, 1131-1144.

Grahn, E., Stonehouse, N.J., Adams, C.J., Fridborg, K., Beigelman, L., Matulic-Adamic, J., Warriner, S.L., Stockley, P.G. and Liljas, L. (2000) "Deletion of a single hydrogen bonding atom from the MS2 RNA operator leads to dramatic rearrangements at the RNA-coat protein interface." NAR 28, 4611-4616.

Funding

We acknowledge the support of the BBSRC and Leverhulme.