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 proposed to be important for translational autoregulation. The thermal denaturation of three in vitro transcribed RNAs corresponding to the pseudoknot region has been investigated as a function of Mg2+ concentration to begin to elucidate the determinants of the structure and stability 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, termed T4-32, contains the native sequence form U(-67) to C(40) with 5'GC and 5'CA single-stranded regions appended to the 5' and 3' ends of the core sequence, respectively. T4-28 contains only the 28 core nucleotides, 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 single major melting transition over a wide range of [Mg2+] (0-6 mM). The delta H degree of unfolding for T4-35 and T4-32 shows a large dependence on Mg2+ concentration; the maximum delta H degree occurs at about 2.0 mM Mg2+ with further addition of Mg2+ simply increasing the tm. Investigation of the [Mg2+] dependence of the tm 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 degree (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 sequence molecule. In contrast to this behavior, T4-28 gives consistently higher tm'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 about 2-3 kcal mol(-1) relative to T4-35, depending upon cation concentration. (1)H NMR experiments suggest that, even in the presence of 4.0 mM Mg2+, T4-28 RNA does not adopt a stable pseudoknotted conformation. These data show that the stability of the pseudoknot in the gene 32 mRNA encoded by the 28-nucleotide core sequence is significantly influenced by the number and nature of the immediately adjacent "single-stranded" 5' and/or 3' nucleotides appended to the core structure. These findings are discussed within the context of the structural model for the evolutionarily related phage T2 and T6 gene 32 mRNA pseudoknots presented in the following paper [Du, Z., Giedroc, D. P., & Hoffman, D. W. (1996) Biochemistry 35, 4187-4198].