THE QUATERNARY GEOMETRY OF TRANSCRIPTION TERMINATION FACTOR-RHO - ASSIGNMENT BY CHEMICAL CROSS-LINKING

Transcription termination factor rho from Escherichia coli is a ring-s haped homohexamer of 419 amino acid subunits and catalyzes an ATP-depe ndent release of nascent RNA transcripts. Previous chemical cross-link ing studies suggested that the rho hexamer might have D-3 symmetry wit h three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F-1-ATPa se rather argued for C-6 symmetry. To resolve this discrepancy, we hav e re-investigated the pattern of cross-linking of rho using various cr oss-linkers with different functional groups and spacer lengths. Upon reaction with dimethyl suberimidate followed by SDS-polyacrylamide gel electrophoresis, rho protein generated a series of cross-linked oligo mers up to hexamers, of which dimers migrated as distinct doublet band s of approximately equal intensities. However, the lower band became m uch stronger than the upper one with dimethyl adipimidate and difluoro dinitrobenzene, and vice verse with disuccinimidyl glutarate, disuccin imidyl suberate and disultosuccinimidyl tartarate. Furthermore, the tr imeric products also produced doublet bands, whose relative intensitie s were again variable with cross-linkers, but in an inverse correlatio n with those of the dimer bands. These results combined with theoretic al considerations support a C-6 symmetry model in which cross-linking is assumed to occur stochastically at one of two alternative sites wit hin each subunit interface with variable relative frequencies dependin g on cross-linkers. The D-3 symmetry is excluded, for the putative tri meric subspecies should always retain mutually equal intensities in th at case. Detailed inspections of the cross-linking kinetics further re vealed a moderate characteristics of C-3 symmetry for the rho hexamer such that the collective as well as relative rates of cross-linking at the two available sites could fluctuate between alternating interface s. The final model designated as C-3/6 is also compatible with other f unctional and structural properties known for rho. (C) 1997 Academic P ress Limited.