Structure of the Autoregulatory Pseudoknot within the Gene 32 Messenger RNA of Bacteriophages T2 and T6:  A Model for a Possible Family of Structurally Related RNA Pseudoknots

A 36-nucleotide RNA with a sequence corresponding to the 5‘ end region of the gene 32 mRNA of bacteriophages T2 and T6 was analyzed by one- and two-dimensional NMR methods. NMR results provide clear evidence that the RNA is folded into a pseudoknot structure with two coaxial stems connected by two loops, in a classic pseudoknot topology. The pseudoknot is unusual in that one of the loops consists of only one nucleotide, which spans the major groove of a seven base pair helical stem. Imino proton resonances indicate the hydrogen bonding pattern within the pseudoknot, and two-dimensional NOE spectra provide information that describes many of the structural features. The temperature dependence of the UV absorption and imino proton exchange rates provides insight into the stability of the pseudoknot. A three-dimensional model of the pseudoknot that is consistent with our NMR data is presented, and features that may be important for stabilizing the pseudoknot structure are discussed. A substantial number of other putative RNA pseudoknots described in the literature have sequences and topologies that appear to be related to the T2 and T6 pseudoknots. We propose that these RNAs may be members of a family of pseudoknots related by a similar structural motif, which we refer to as “common pseudoknot motif 1” or CPK1. The bacteriophage T2/T6 pseudoknot can be considered a structural model for the CPK1 family. The common features of the CPK1 pseudoknots are a stem 2 with six or seven base pairs, a loop 1 consisting of a single adenosine, and a variable length stem 1 and loop 2. The first “dangling” nucleotide at the 3‘ end of the molecule probably stabilizes stem 2. The CPK1 family includes several of the retroviral pseudoknots associated with mRNA frameshifting and readthrough. The work presented here describes the first detailed NMR analysis of an RNA pseudoknot with an entirely natural nucleotide sequence.