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 within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta 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 delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 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 delta 549 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 replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood 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 first time.

Translational activation in coliphage Qbeta: on a polycistronic messenger RNA, repression of one gene can activate translation of another.

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qbeta maturation gene remains translationally "off" by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched "on" when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

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 analysis of the bacteriophage Q beta readthrough cistron. In the Q beta cDNA-containing plasmid, pQ beta m100, we constructed six defined Q beta deletion cDNA genomes, each missing between 86 and 447 nucleotides from within the readthrough cistron. These deletion plasmids were introduced into host cells that are constitutively supplied with Q beta readthrough 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 potential. Isolated readthrough-defective phage particles were subsequently used to infect host cells that carried helper readthrough protein. Passaged viruses yielded both larger plaques and higher titers, compared with those of the parent phages. Sequence analysis revealed that the genomes of the passaged viruses had deleted additional regions of readthrough RNA sequence. We discuss the possibilities that (1) the disruption of a well-defined structural domain in Q beta RNA was selectively disadvantageous to phage infection, and that (2) the evolved viral populations were selected by virtue of their ability to restore critical integrity of short and/or long-range nucleotide interactions within this region of Q beta RNA.

A complete plasmid-based complementation system for RNA coliphage Q beta: three proteins of bacteriophages Q beta (group III) and SP (group IV) can be interchanged.

Our laboratory has established a bacteriophage Q beta cDNA-containing plasmid system in which virtually all coding defects present within the 4217 nucleotide Q beta genome can be complemented in trans. In this system, Q beta minus strand RNAs are constitutively transcribed from plasmid cDNA by Escherichia coli RNA polymerase. Replication of these minus strands results in the synthesis of Q beta plus RNA, thereby triggering an infectious cycle in which Q beta phase particles are generated. Genetically engineered Q beta genome mutations that result in defective viral proteins can be complemented in trans by the products of one or more Q beta helper plasmids that express either: (1) Q beta maturation protein, which can complement defects in the Q beta maturation cistron (nucleotides 61 to 1320); (2) Q beta readthrough protein, which can complement defects in the readthrough cistron (nucleotides 1344 to 2330); or (3) Q beta replicase, which can complement defects in the replicase cistron (nucleotides 2352 to 4118). Each plasmid component of this system contains a unique origin of replication and carries a different antibiotic gene, thereby enabling all combinations of these plasmids to coexist in the same host. We have further developed a second series of helper plasmids that generate the corresponding viral proteins of the related group IV RNA phage SP. Each of these SP helper proteins can complement respective defects within the Q beta genome with efficiencies similar to those observed for the Q beta helper proteins. It is now possible to supply functional Q beta or SP proteins in trans to examine Q beta genomes that contain protein coding defects for their ability to synthesize Q beta proteins, replicate Q beta RNA, assemble virions, and/or lyse the host cell.

Coliphage Q beta RNA replication: RNA catalytic for single-strand release.

We have generated 14 recombinant RNA templates for Q beta replicase, each having either an exogenous inverted repeat sequence or a sequence with no repeat. These templates were used to initiate in vitro replication by Q beta replicase in amounts that saturated the enzyme. We observed that replication rates for RNAs that putatively contained secondary structures in the recombinant sequences ranged from 33 to 69% that of a wild-type MDV-1 RNA control, regardless of the size of the inserted hairpin. Moreover, most of the newly synthesized RNA was present as single strands. Alternatively, RNAs that contained exogenous sequences not expected to form secondary structures exhibited replication rates less than 25% that of MDV-1. In each case, the reaction rate was correlated with the length of the insertion, and the majority of product RNA consisted of duplexed molecules (complementary plus and minus strands hybridized together). When these same recombinant RNAs were used in reactions in which the molar amount of RNA template was 10(6)-10(7) times lower than that of the replicase, only those that putatively contained secondary structures survived in the replication reaction. Our results are consistent with the theory that hairpin structure formation during RNA synthesis by Q beta replicase directly influences the regeneration of single-stranded RNA products.

Q beta RNA bacteriophage: mapping cis-acting elements within an RNA genome.

We have identified, for the first time, regions of cis-acting RNA elements within the bacteriophage Q beta replicase cistron by analyzing the infectivities of 76 replicase gene mutant phages in the presence of a helper replicase. Two separate classes of mutant Q beta phage genomes (35 different insertion mutants, each containing an insertion of 3 to 15 nucleotides within the replicase gene, and 41 deletion genomes, each having from 15 to 935 nucleotides deleted from different regions of the gene) were constructed, and their corresponding RNAs were tested for the ability to direct the formation of progeny virus particles. Each mutant phage was tested for plaque formation in an Escherichia coli (F+) host strain that supplied helper Q beta replicase in trans from a plasmid DNA. Of the 76 mutant genomes, 34% were able to direct virus production at or close to wild-type levels (with plaque yield ratios of greater than 0.5), another 36% also produced virus particles, but at much lower levels than those of wild-type virus (with plaque yield ratios of less than 0.05), and the remaining 30% produced no virus at all. From these data, we have been able to define regions within the Q beta replicase gene that contain functional cis-acting RNA elements and further correlate them with regions of RNA that are solely required to code for functional RNA polymerase.

Q beta replicase: mapping the functional domains of an RNA-dependent RNA polymerase.

We have localized a functional region of the RNA bacteriophage Q beta replicase following an extensive mutational analysis. Using the method of oligonucleotide linker-insertion mutagenesis, we specifically introduced mutations into a cloned DNA copy of the Q beta replicase gene so that the resulting replicase products would putatively contain small amino acid insertions. In a selective phenotypic assay, we screened mutant replicases for RNA-directed replication activity in vivo. Analysis of 37 different mutant clones indicated that Q beta replicase can accept amino acid substitutions and insertions at several sites at the amino and carboxy termini without abolishing functional activity in vivo or in vitro. However, disruption within the internal amino acid sequence resulted almost exclusively in nonfunctional enzyme. The results suggest that the central region of the replicase protein contains a rigid amino acid composition that is required for replicase function, whereas the amino and carboxy termini are much more receptive to small amino acid insertions and substitutions. These experiments should further enable us to analyze the coding function of the Q beta replicase gene independently of other phage RNA functions contained within this nucleotide region.