STRUCTURAL PLASTICITY IN RNA AND ITS ROLE IN THE REGULATION OF PROTEIN TRANSLATION IN COLIPHAGE Q-BETA

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (Delta 159 and Delta 549) located withi n the read-through domain, a 850 nucleotide hairpin, in coliphage Q be ta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral geno mes carrying deletion Delta 159. Translational regulation is restored in deletion Delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within t he read-through domain of Delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicte d structure in the readthrough domain of evolved pseudorevertant Delta 549 is unusually well determined. Structural analysis by electron mic roscopy of the genomic RNAs shows that several long range helices at t he base of the readthrough domain, that suppress translational initiat ion of the viral replicase gene in the wild-type genome, have been des tabilized in Delta 159 RNA. In addition, the structure of local hairpi ns within the read-through region is more variable in Delta 159 RNA th an in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of Delta 549 RNA. Our analyses suggest that struc ture throughout the read-through domain affects the regulation of vira l replicase expression by altering the likelihood that long-range inte ractions at the base of the domain will form. We discuss possible kine tic and equilibrium models that can explain this effect, and argue tha t observed changes in structural plasticity within the read-through do main of the mutant genomes are key in understanding the process. Durin g the course of these studies, we became aware of the importance of th e information contained in the energy dot plot produced by the RNA sec ondary structure prediction program mfold. As a result, we have improv ed the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is pres ented here for the first time. (C) 1998 Academic Press Limited.