THERMODYNAMICS OF FOLDING OF THE RNA PSEUDOKNOT OF THE T4 GENE-32 AUTOREGULATORY MESSENGER-RNA

Nucleotides U(-67) to C(-40) at the extreme 5' end of the gene 32 mRNA in bacteriophage T4 have been shown to fold into an RNA pseudoknot pr oposed to be important for translational autoregulation. The thermal d enaturation of three in vitro transcribed RNAs corresponding to the ps eudoknot region has been investigated as a function of Mg2+ concentrat ion to begin to elucidate the determinants of the structure and stabil ity of this conformation. T4-35 is a 35-nucleotide RNA containing a 5' G followed by the natural T4 sequence starting with the mature 5' end of the mRNA, nucleotides A(-71) to C(-38). A 32-nucleotide RNA, terme d T4-32, contains the native sequence from U(-67) to C(-40) with 5'-GC and 5'-CA single-stranded regions appended to the 5' and 3' ends of t he core sequence, respectively. T4-28 contains only the 28 core nucleo tides, and the predicted closing U(-67)-A(-52) base pair in stem 1 has been replaced with a phylogenetically allowed G(-67)-C(-52) base pair , Ribonuclease mapping of T4-32 and imino proton NMR experiments of T4 -35 show that both sequences adopt a pseudoknotted conformation. At pH 6.9 and 50 mM NaCl, T4-35 and T4-32 RNAs are characterized by a singl e major melting transition over a wide range of [Mg2+] (0-6 mM). The D elta H degrees of unfolding for T4-35 and T4-32 shows a large dependen ce on Mg2+ concentration; the maximum Delta H degrees occurs at about 2.0 mM Mg2+ with further addition of Mg2+ simply increasing the t(m). Investigation of the [Mg2+] dependence of the t(m) suggests that a net of one Mg2+ ion is released upon denaturation of T4-35 and T4-32 RNAs . Over the entire [Mg2+] range, the Delta G degrees (37 degrees C) for the folding of T4-35 is consistently 1-1.5 kcal mol(-1) more negative than T4-32 due to a higher stabilization enthalpy for the natural seq uence molecule, In contrast to this behavior, T4-28 gives consistently higher t(m)'s but less negative enthalpies and is destabilized (at 37 degrees C) by about 0.5-1.5 kcal mol(-1) relative to T4-32 and by abo ut 2-3 kcal mol(-1) relative to T4-35, depending upon cation concentra tion. H-1 NMR experiments suggest that, even in the presence of 4.0 mM Mg2+, T4-28 RNA does not adopt a stable pseudoknotted conformation. T hese data show that the stability of the pseudoknot in the gene 32 mRN A encoded by the 28-nucleotide core sequence is significantly influenc ed by the number and nature of the immediately adjacent ''single-stran ded'' 5' and/or 3' nucleotides appended to the core structure. These f indings are discussed within the context of the structural model for t he evolutionarily related phage T2 and T6 gene 32 mRNA pseudoknots pre sented in the following paper [Du, Z., Griedroc, D. P., & Hoffman, D. W. (1996) Biochemistry 35, 4187-4198].