Molecular interactions in the RNA bacteriophage MS2.

The simple RNA bacteriophages of E.coli have long been used as ideal model systems in which the details of RNA replication, translation and the control of gene expression can be studied in molecular detail’.’. In particular, the translational repression of the phage replicase cistron by coat protein subunits has attracted a great deal of attention and this has become a paradigm for sequence-specific RNA-protein interactions. In a series of pioneering experiments, Uhlenbeck and his colleagues were able to demonstrate that the key recognition determinants in the RNA lay entirely within a 19 nucleotide (nt) fragment (TR) capable of forming a ~ t e m l o o p ~ ~ . Further experiments suggested that this interaction was also the trigger of phage assembly in vitro and in vivo ’,’. Thus the specific interaction between the RNA and the phage coat protein subunits is used to regulate two distinct aspects of the phage life-cycle, translational repression of the replicase and assembly initiation. Recently, the molecular basis of the sequence-specific interaction has been revealed by determination of the X-ray crystal structure of a C-5-variant TR-coat protein complex at 3.0 8, resolution’. Both C S-variant and wild-type TR complexes have now been refined at 2.7 A (Valegird et al., in progress). These complexes were prepared by soaking crystals of RNA-free T=3 capsids with TR fragments which were able to penetrate to the centre of the particles, presumably via the channels at the particle five-fold and/or three-fold axes. Each T=3 capsid consists of 60 coat protein dimers in the AIB conformation and 30 coat protein dimers in the C/C conformation. At saturating levels of TR RNA, each coat protein dimer within the capsid became bound to an RNA molecule. Perhaps surprisingly, the RNA bound in a unique orientation at the A/B protein dimer positions allowing the details of the protein-RNA interaction to be seen for the first time. Experiments with chemical variants of the TR sequence suggest that the complex which forms in solution between a coat protein dimer and the TR is the same, or very similar to, the one seen in the crystal structure of the operator c a p s i d ~ ‘ ~ ’ ~ . The unique orientation of the TR-coat protein A/B dimer interaction suggests that coat protein conformation and RNA affinity may be coupled. The crystal structures of operator capsids containing subfragments of the TR sequence are also consistent with this view (Valeghd et al., in progress). In v i m , RNA-free MS2 capsids can be reassembled from dissociated coat protein dimers (subunit Mr 13.75 kDa) into fully formed capsids (M, 2.5 MDa) by a simple pH jump from 3.8 to 7.4I5.I6. These reassembled capsids are morphologically and hydrodynamically indistinguishable from native phage. Coat protein reassembly in the absence of RNA, results in a small (-5%) decrease in the total tryptophan intrinsic fluorescence emission intensity, probably reflecting changes in solvent accessibility of the tryptophan residues (W32 & W82). Kinetic analysis suggests that reassembly is dominated by a second-order process consistent with the addition of coat protein dimers to a growing phage shell. Experiments at different ionic strengths imply that charge neutralisation plays little part in driving this reassembly reaction. Addition of TR RNA at this stage results in little change to the apparent rate of reassembly. Pre-incubation of the dissociated coat protein subunits with the TR fragment at low pH, however, results in a dramatic increase in the yield of capsids and, consequently, also in the amplitude of the fluorescence change, without significant changes to the overall rate. Reassembly reactions in the presence of tRNA, as a control, are similar to protein-alone reactions, suggesting that reassembly after equilibration with TR is stimulated by sequence-specific binding. RNA titrations show that this effect reaches a maximum at a sub-stoichiometric molar ratio of TR RNA:coat protein dimer. These data suggest that RNA-binding stabilises a quasi-equivalent protein dimer conformation, which acts as the assembly initiator. The extreme co-operativity of the subsequent reassembly reaction can, however, only be accounted for by the formation of a subsequent nucleation complex containing at least five coat protein dimers, equivalent to formation of a pentamer of the T=3 capsid. There are no kinetically significant intermediates between the nucleation complex and the final capsid.