Li H, Dalal S, Kohler J, Vilardell J, White S A
Department of Chemistry, Bryn Mawr College, PA 19010, USA.
J Mol Biol. 1995 Jul 21;250(4):447-59. doi: 10.1006/jmbi.1995.0389.
The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5' end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G-U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.
利用化学和酶促探针以及热力学方法研究了酿酒酵母核糖体蛋白L32的RNA结合靶标的结构。在体内，酵母RPL32的产生受一种反馈机制调节，即RPL32与其转录本的5'端结合并抑制剪接。核糖体蛋白L32在L32 RNA转录本上的结合位点可缩减至不到30个核苷酸，这些核苷酸构成了一个茎-内环-茎结构基序。内环由一个潜在的G-U碱基对封闭，不对称且主要包含嘌呤。通过化学和酶促探针证实了两个螺旋区域的存在。环区域的反应性表明其结构介于单链和双链RNA之间。碱基堆积延伸至环中，但有两个环碱基对化学试剂极具反应性。模型RNA与蛋白质之间的相互作用具有特异性，解离常数约为10 nM。如突变数据以及化学保护和修饰干扰研究所表明的，几个环碱基对蛋白质结合至关重要。内环使RNA不稳定，并使RNA以全或无的方式解链。
