Theimer C A, Wang Y, Hoffman D W, Krisch H M, Giedroc D P
Department of Biochemistry and Biophysics, Texas A&M University, Cóllege Station 77843-2128, USA.
J Mol Biol. 1998 Jun 12;279(3):545-64. doi: 10.1006/jmbi.1998.1812.
The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-even and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot. In phage T4, the pseudoknot is contained within 28 contiguous nucleotides which adopt a pseudocontinuous helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequivalent single-stranded loops of five and one nucleotide(s). These two loops cross the minor and major grooves of stems 1 and 2, respectively. In this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T4 gene 32 mRNA has been determined through analysis of dual-wave-length, equilibrium thermal melting profiles via application of a van't Hoff model based on multiple sequential, two-state transitions. The melting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration. Compensatory base-pair substitutions incorporated into helical stems 1 and 2 were used to assign the first low enthalpy, moderate tm melting transition to the denaturation of the short three to four base-pair stem 1, followed by unfolding of the larger seven base-pair stem 2. We find that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) strikingly uncouple the melting of stems 1 and 2, with the U10 substitution and the GA10 loop expansion more destabilizing than the G10 and C10 substitutions. A significant increase in the extent of cleavage by RNase T1 following the conserved G26 (the 3' nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix junction region in this mutant may be responsible, at least in part, for this uncoupling. In addition to a modest destabilization of stem 2, the major effect of deletion or nucleotide substitution in the 3' single-stranded tail is a destabilization of stem 1, a non-nearest neighbor tertiary structural effect, which may well be transmitted through an altered loop 1-core helix interaction. In contrast, truncation of the 5' tail has no effect on the stability of the molecule.
17种T偶数及相关噬菌体的基因32的上游自调控mRNA前导序列折叠成一种简单的三级结构基序，即发夹型RNA假结。在噬菌体T4中，假结包含在28个连续的核苷酸内，这些核苷酸形成一种假连续螺旋结构，该结构源自由四个（茎1）和七个（茎2）碱基对组成的两个同轴堆叠的螺旋茎，并由五个和一个核苷酸的两个不等价单链环连接。这两个环分别穿过茎1和茎2的小沟和大沟。在本研究中，通过应用基于多个连续双态转变的范特霍夫模型，对双波长平衡热解链曲线进行分析，确定了与T4基因32 mRNA的野生型和序列变体相对应的35个核苷酸RNA片段的平衡解折叠途径。在很宽的镁离子浓度范围内，野生型RNA的解链曲线可以很好地用两个连续的解链转变来描述。将补偿性碱基对替换引入螺旋茎1和茎2，以将第一个低焓、中等解链温度的解链转变归因于短的三到四个碱基对的茎1的变性，随后是较大的七个碱基对的茎2的解折叠。我们发现，环1替换突变体（A10突变为G10、C10、U10或GA10）显著地使茎1和茎2的解链解偶联, 其中U10替换和GA10环扩展比G10和C10替换更能使结构不稳定。在U10、G10和GA10突变体中，保守的G26（环2中的3'核苷酸）之后，RNase T1切割程度显著增加，这表明该突变体中改变的螺旋-螺旋连接区域可能至少部分地导致了这种解偶联。除了茎2有适度的不稳定外，3'单链尾部的缺失或核苷酸替换的主要影响是茎1的不稳定，这是一种非紧邻三级结构效应，很可能是通过改变的环1-核心螺旋相互作用传递的。相比之下，5'尾部的截短对分子稳定性没有影响。
