Pseudoknot Structures in Retroviral and Bacteriophage Messenger RNA: a Family of Structurally Related RNA Pseudoknots

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three-dimensional structure of an RNA pseudoknot with a sequence corresponding to the 5' end region of the gene 32 messenger RNA of bacteriophage T2. NMR results show that the pseudoknot contains two coaxial A-form helical stems connected by two loops. One of the loops consists of a single nucleotide, which spans the major groove of the seven base pair helical stem 2. The second loop consists of 7 nucleotides, and spans the minor groove of stem 1. A three-dimensional model of the pseudoknot that is consistent with the NMR data will be presented, and features that are likely to be important for stabilizing the pseudoknot structure will be described.A combination of NMR and phylogenetic methods were used to characterize the structural features of RNA pseudoknots that are associated with frameshift and readthrough sites within the retroviral gag-pro messenger RNA. The majority of the retroviral frameshift and readthrough sites were found to be followed by nucleotide sequences that have the potential to form pseudoknots with structures that are remarkably similar to that of the bacteriophage T2 gene 32 mRNA.

An NMR and Mutational Study of the Pseudoknot Within the Gene 32 mRNA of Bacteriophage T2: Insights into a Family of Structurally Related RNA Pseudoknots

NMR methods were used to investigate a series of mutants of the pseudoknot within the gene 32messenger RNA of bacteriophage T2, for the purpose of investigating the range of sequences, stem and loop lengths that can form a similar pseudoknot structure. This information is of particular relevance since the T2 pseudoknot has been considered a representative of a large family of RNA pseudoknots related by a common structural motif, previously referred to as ‘common pseudoknot motif 1’ or CPK1. In the work presented here, a mutated sequence with the potential to form a pseudoknot with a 6 bp stem2 was shown to adopt a pseudoknot structure similar to that of the wild-type sequence. This result is significant in that it demonstrates that pseudoknots with 6 bp in stem2 and a single nucleotide in loopl are indeed feasible. Mutated sequences with the potential to form pseudoknots with either 5 or 8 bp in stem2 yielded NMR spectra that could not confirm the formation of a pseudoknot structure. Replacing the adenosine nucleotide in loopl of the wild-type pseudoknot with any one of G, C or U did not significantly alter the pseudoknot structure. Taken together, the results of this study provide support for the existence of a family of similarly structured pseudoknots with two coaxially stacked stems, either 6 or 7 bp in stem2, and a single nucleotide in loop1. This family includes many of the pseudo-knots predicted to occur downstream of the frameshift or readthrough sites in a significant number of viral RNAs.

Direct stereoscopic visualization of icosahedral capsid geometry in bacteriophage T4 freeze-fractured, deep-etched replicas

Bacteriophage T4 is one of the most complex of the tailed bacteriophage. Its DNA is packaged inside a protein capsid whose structure has long been the subject of study. All T-even bacteriophage appear to have a capsid structure based upon an icosahedral geometry. This has been established indirectly by a combination of symmetry considerations and physical and biochemical experiments, including some electron microscopy. Perhaps the most persuasive demonstration of the icosahedral capsid geometry in bacteriophage T2 is not based on a direct visualisation of the T2 capsid. Instead, the argument relies critically on the authors' ability to accurately count protein capsomers on the T2 capsid replica of a freeze-fractured, etched sample. Moreover, the icosahedral geometry of T4 capsids is simply inferred from results on T2. In the present study, we visualize directly the prolate icosahedral capsid geometry of T4 bacteriophage from stereopairs of micrographs of replicas produced by the freeze-fracture, deep-etch technique utilizing vertical replication to identify the triangular faces comprising the icosahedral T4 capsid.