Vartikar J V, Draper D E
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
J Mol Biol. 1989 Sep 20;209(2):221-34. doi: 10.1016/0022-2836(89)90274-x.
The region of the Escherichia coli 16 S ribosomal RNA recognized by the ribosomal protein S4 has been defined by assaying a set of 13 16 S rRNA fragments for S4 binding. The fragments were prepared by transcription in vitro, and binding constants were measured in three ways: retention of labeled RNA fragments on nitrocellulose filters by S4; co-sedimentation of labeled S4 with RNA fragments in sucrose gradients; and the distribution of labeled S4 between two RNAs of different sizes in a sucrose gradient. All three methods gave similar relative binding strengths for a variety of 16 S rRNA and non-specific (23 S rRNA) sequences, with the exception of two of the largest 16 S rRNA fragments; these gave smaller association constants in the filter retention assay than in the other methods. We found that specific complexes of S4 with these larger RNAs do not bind well to filters, leaving non-specific complexes to dominate the assay. Specific complexes with RNAs less than or equal to 891 nucleotides were retained efficiently by S4 on filters, and gave reliable binding constants. All 16 S rRNA fragments containing nucleotides 39 to 500 bound S4 with the same affinity as intact 16 S rRNA, while all fragments with endpoints within 39 to 500 bound at least tenfold more weakly. This sequence must be able to fold independently of the rest of the rRNA. Comparison of this minimal 462-nucleotide S4 binding site with S4 footprinting results suggests that S4 binding might alter the conformations of RNA neighboring the 39 to 500 region in the intact 16 S rRNA. Specific S4-rRNA binding is not sensitive to KCl concentration, but a more normal salt dependence is seen in K2SO4 (delta logK/delta log[K+] approximately -3.3). This duplicates the behavior of the specific S4-alpha mRNA translational repression complex, arguing that S4 recognizes both the mRNA and rRNA substrates by the same mechanism. Mg2+ is not required to form the specific rRNA complex, at least under conditions which stabilize RNA structure (0.35 M-KCl, 5 degrees C).
通过检测一组13个16S rRNA片段与核糖体蛋白S4的结合，确定了大肠杆菌16S核糖体RNA中被核糖体蛋白S4识别的区域。这些片段通过体外转录制备，结合常数通过三种方法测量：标记的RNA片段被S4保留在硝酸纤维素滤膜上；标记的S4与RNA片段在蔗糖梯度中共沉降；以及标记的S4在蔗糖梯度中在两种不同大小的RNA之间的分布。除了两个最大的16S rRNA片段外，所有这三种方法对各种16S rRNA和非特异性（23S rRNA）序列都给出了相似的相对结合强度；在滤膜保留试验中，这两个片段给出的缔合常数比其他方法中的小。我们发现S4与这些较大RNA的特异性复合物不能很好地结合到滤膜上，导致非特异性复合物在试验中占主导。S4能有效地将与小于或等于891个核苷酸的RNA形成的特异性复合物保留在滤膜上，并给出可靠的结合常数。所有包含核苷酸39至500的16S rRNA片段与完整的16S rRNA以相同的亲和力结合S4，而所有端点在39至500范围内的片段结合强度至少弱十倍。该序列必须能够独立于rRNA的其余部分折叠。将这个最小的462个核苷酸的S4结合位点与S4足迹分析结果进行比较表明，S4结合可能会改变完整16S rRNA中39至500区域附近RNA的构象。特异性的S4-rRNA结合对KCl浓度不敏感，但在K2SO4中观察到更正常的盐依赖性（δlogK/δlog[K+]约为-3.3）。这与特异性的S4-α mRNA翻译抑制复合物的行为一致，表明S4通过相同的机制识别mRNA和rRNA底物。至少在稳定RNA结构的条件下（0.35 M-KCl，5℃），形成特异性rRNA复合物不需要Mg2+。
