Structural Plasticity in RNA and its Role in the Regulation of Protein Translation in Coliphage Qb A. B. Jacobson 1 *, R. Arora 2 , M. Zuker 3 , C. Priano 2 , C. H. Lin 1 and D. R. Mills 2 1 Department of Microbiology State University of New York Stony Brook, NY 11794-5222, USA 2 Department of Microbiology and Immunology Downstate Medical Center SUNY at Brooklyn NY 11203-2098, USA 3 Institute for Biomedical Computing, Washington University, St. Louis MO 63110, USA We have analyzed both conformational and functional changes caused by two large cis-acting deletions (Á159 and Á549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Qb genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion Á159. Translational regulation is restored in deletion Á549, a naturally evolved pseudorevertant. Structural analysis by computer mod- eling shows that structural features within the read-through domain of Á159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read- through domain of evolved pseudorevertant Á549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read- through domain, that suppress translational initiation of the viral repli- case gene in the wild-type genome, have been destabilized in Á159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in Á159 RNA than in wild-type RNA. Stable RNA sec- ondary structure is restored in the read-through domain of Á549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likeli- hood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the ®rst time. # 1998 Academic Press Limited Keywords: Qb; RNA structure; translational regulation; annotated structure plots; electron microscopy *Corresponding author Introduction We have been studying the properties and func- tion of large, unusually stable structural features that are observed by electron microscopy, using the basic protein ®lm technique (Davis et al., 1971), in genomic RNAs of the RNA coliphages (Jacobson et al., 1988; Jacobson, 1991; Skripkin & Jacobson, 1993). These features range in size from 150 to 1500 nt and nearly all of them are located within coding regions of the viral genome. To study their function, we are using a genetic system that was developed for RNA coliphage Qb, in which viral proteins are supplied in trans on independent plas- mids (Arora, 1995; Priano et al., 1995). Cells carry- ing these plasmids are transformed with cloned cDNA. Cells transformed with wild-type cDNA produce viral plaques when plated on a lawn of F  bacterial cells. Viral plaques are also obtained with mutant cDNAs if wild-type protein that is supplied in trans from an independent plasmid can comp- lement the defect. Mutants that are not comple- J. Mol. Biol. (1998) 275, 589±600 0022±2836/98/040589±12 $25.00/0/mb971472 # 1998 Academic Press Limited