Département de Biochimie, Université de Montréal, Montréal, CP 6128, Succursale Centre-Ville, QC H3C 3J7, Canada.
Nucleic Acids Res. 2010 Jun;38(10):3441-53. doi: 10.1093/nar/gkq031. Epub 2010 Feb 5.
To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.
为了理解核糖体 RNA 的核苷酸序列如何决定其三级结构，我们开发了一种新的方法来鉴定 rRNA 序列中负责形成不同短程和长程相互作用的特征。该方法基于对特定重复 RNA 基序的几个实例的共同分析。对于基序的不同情况，我们设计了组合基因文库，其中等价核苷酸位置被随机化。通过设计文库的体内表达，我们选择那些提供功能性核糖体的变体。然后，对所选克隆的核苷酸序列进行分析，就可以确定对基序的每个情况施加的序列约束。所有情况下共有的约束被解释为提供了基序的完整性，而那些特定于个别情况的约束则使基序能够适应特定的结构背景。在这里，我们通过三个所谓的沿沟堆积基序的实例证明了这种方法的有效性，这些基序存在于核糖体 RNA 的不同部分。
