Powers T, Daubresse G, Noller H F
Sinsheimer Laboratories, University of California, Santa Cruz 95064.
J Mol Biol. 1993 Jul 20;232(2):362-74. doi: 10.1006/jmbi.1993.1396.
One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly. As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly. One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0 degrees C effects). At 30 degrees C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2 = < 5 min at 30 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees C). Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly. These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5' to 3' polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3' major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late-assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.
核糖体领域尚未解决的重要问题之一是核糖体组装的顺序性和协同性的分子基础。作为解决这个问题的一种方法，我们利用了体外重组对温度的依赖性，并使用化学探测方法来检查亚基组装过程中连续阶段16 S rRNA的构象和反应活性。当RNA和蛋白质在没有任何加热步骤（0摄氏度效应）的情况下孵育时，一类核苷酸显示出与天然30 S颗粒中观察到的反应活性相似。在30摄氏度时，组装过程需要2小时，其他碱基可归为几个额外的动力学类别之一，这取决于它们的化学反应活性从裸RNA中观察到的水平转变为完全组装亚基中观察到的水平的速率：（1）快速（30摄氏度下t1/2 = < 5分钟）；（2）缓慢（30摄氏度下t1/2 = 15至30分钟）；（3）延迟缓慢（30摄氏度下t1/2 = 30至60分钟）。最后，几个核苷酸在其反应活性上表现出瞬态动力学，在早期时间点显示出增加的反应活性，而在组装后期变得受到保护；这些效应大多对应于先前在逐步体外组装过程中显示出相互增强和保护模式的残基。这些发现，连同我们之前使用纯化的单个蛋白质进行的研究，得出以下结论：（1）体外组装存在从5'到3'的主要极性，尽管它与转录解偶联；（2）中央和3'主要结构域的部分仅在组装的非常后期折叠成活性构象；（3）根据30 S亚基组装图谱，由后期组装蛋白质覆盖的碱基通常比在组装图谱早期结合的蛋白质覆盖的残基显示出更慢的动力学，为体外组装过程的顺序性提供了直接证据；（4）大多数蛋白质与属于多个动力学类别的核苷酸相关，这表明组装通过多种途径进行，或者单个蛋白质与RNA的不同区域顺序相互作用。
