ANALYSIS OF RNA PHAGE FR COAT PROTEIN ASSEMBLY BY INSERTION, DELETIONAND SUBSTITUTION MUTAGENESIS

A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, dele tion and substitution mutagenesis. Mutations were specifically introdu ced into either pre-existing or artificially created restriction enzym e sites within fr CP gene expressed in Escherichia coli from a recombi nant plasmid. This directs synthesis of wild type protein that undergo es self-assembly and forms capsid-like particles indistinguishable mor phologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions whic h are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their asse mbly properties investigated. The majority of mutants demonstrated red uction of assembly ability and formed either CP dimers (mutations at r esidues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A thi rd type of assembled structure was formed by a variant with a single a mino acid substitution I104T. The alphaA-helix region (residues 97-111 ) is particularly sensitive to mutation and any alteration in this reg ion decreases accumulation of mutant protein in E.coli. The relative c ontributions of particular fr CP domains in maintenance of capsid stru ctural integrity as well as the possible capsid assembly mechanism are discussed.