CIS-ACTING ELEMENTS WITHIN AN RNA COLIPHAGE GENOME - FOLD AS YOU PLEASE, BUT FOLD YOU MUST

Using an in vivo complementation system, we conducted a mutational ana lysis of the bacteriophage Q beta readthrough cistron. In the Q beta c DNA-containing plasmid, pQ beta m100, we constructed six defined Q bet a deletion cDNA genomes, each missing between 86 and 447 nucleotides f rom within the readthrough cistron. These deletion plasmids were intro duced into host cells that are constitutively supplied with Q beta rea dthrough protein from the plasmid pQ beta RT. Under these conditions, all six deletion genomes spontaneously generated phage particles, each exhibiting a characteristic plaque phenotype and virus forming potent ial. Isolated readthrough-defective phage particles were subsequently used to infect host cells that carried helper readthrough protein. Pas saged viruses yielded both larger plaques and higher titers, compared with those of the parent phages. Sequence analysis revealed that the g enomes of the passaged viruses had deleted additional regions of readt hrough RNA sequence. We discuss the possibilities that (1) the disrupt ion of a well-defined structural domain in Q beta RNA was selectively disadvantagous to phage infection, and that (2) the evolved viral popu lations were selected by virtue of their ability to restore critical i ntegrity of short and/or long-range nucleotide interactions within thi s region of Q beta RNA. (C) 1996 Academic Press Limited