ARE THE SLOW KINETICS OF CRO DIMER ASSEMBLY CRITICAL TO THE LAMBDA GENETIC SWITCH?

Chemistry and Chemical Engineering

ARE THE SLOW KINETICS OF CRO DIMER ASSEMBLY CRITICAL TO THE LAMBDA GENETIC SWITCH?

John [Satumba.sup.*], Alexander E. Fong, Ibrahim Al- Duraibi, Melva T. James, and Michael C. Mossing University of Mississippi, Oxford, MS 38677-1848

The cro gene is transcribed early but acts late in the genetic program of bacteriophage lambda. Cro binds to its operator DNA as a dimer at nanomolar concentrations but is mostly monomeric in the absence of DNA. The key hydrophobic contacts in the dimer interface are made by residues F58-cis-P59 which form a sharp bend at the end of an intermolecular beta strand. Cro folds rapidly (milliseconds) to a compact intermediate but dimerization requires proline isomerization (hundreds of seconds). The fluorescence intensity of the single tryptophan variant, Cro F58W is sensitive both to the isomeric state of the following peptide bond (cis [less than] trans) and to folding state (folded [greater than] unfolded). Slow rates have all of the characteristics of proline isomerization including the ability to be enhanced by the E. coli PPIase SlyD. Fluorescence resonance energy transfer between W58 and IAEDANS labeled subunits shows that dimerization requires the native proline isomer. Subunit exchange from native dimers is also slow and appears to be limited by dimer dissociation. Population of the monomeric state at low concentrations or in double jump kinetic experiments reveals faster exchange rates. Experiments are underway to investigate the effects of Cro folding variants and PPIase levels on the kinetics of repressor function in vivo.