Escherichia coli RNase P, a tRNA processing enzyme, is composed of M1 RNA and C5 protein. While the role of M1 RNA as the catalytic subunit has been well documented, understanding of the functions of C5 protein as a RNA-binding protein in vivo remains very limited. Interactions of proteins with RNA are important to gene expression and several proteins that bind to RNA are known to be involved in gene regulation. Although C5 protein was originally identified as the protein component of RNase P, it is possible that C5 protein participates in other cellular metabolisms. to understand the function of C5 protein as an RNA binding protein, RNA aptamers binding to C5 protein were selected by SELEX (Systematic Evolution of Ligands by EXponential enrichment) from a library of 108-mers that contained a central variable segment of 42 nucleotides. Sequence analysis of the RNA aptamers suggests that C5 protein can bind to various RNA motifs with comparable dissociation constant to that of M1 RNA. The predominant sequence, named W2, was selected for further study. Interaction between W2 and C5 protein was independent of the $Mg^{2+}$ concentration, which contrasts with the $Mg^{2+}$
-dependency of the M1 RNA-C5 protein interaction. The stability of the ribonucleoprotein of W2-C5 protein was much lower than that of M1 RNA, but it increased with lowering temperatures. The affinity of W2 to C5 protein increased with the concentration of monovalent ion $NH_4^{+}$, suggesting that the interaction occurs through hydrophobic attractions. A core RNA motif essential for interaction with C5 protein was identified as a stem-loop structure composed of the 5-bp stem and 20-nt loop. Many RNA motifs similar to the core RNA motif were present in the E. coli genome sequence. This may imply that C5 protein can function as an interacting partner protein of other cellular RNAs than M1 RNA in in vivo. To identify RNA motifs interacting with C5 protein in vivo, genomic SELEX was carried out with genomic RNA libraries of 120-mers and 150-mers carrying an overlapping set of inserts for every segment of the E coli genome. Various genome-based RNA motifs were selected with their dissociation constants comparable to that of M1 RNA, suggesting that C5 protein can bind to other RNA molecules than M1 RNA in vivo. One of the RNA aptamers, anti-YcaH, which was complementary to the ycaH mRNA, was cleaved by RNase P holoenzyme. The anti-YcaH RNA molecule was not cleaved by M1 RNA alone even in the presence of high concentrations of $Mg^{2+}$. Therefore, C5 protein is essential for cleavage of anti-YcaH by RNase P. The anti-YcaH molecule differs in binding to C5 protein from precursor tRNA molecules that barely bind to C5 protein. The presence of anti-YcaH in vivo was confirmed by Northern analysis although its biological function relevant to RNase P cleavage remains to be demonstrated. The function of C5 protein on the RNase P catalysis was also examined with a precursor E. coli $tRNA^{Phe}$ having a single mismatch in the acceptor stem. This mutant precursor unexpectedly generated upstream cleavage products at the -8 position as well as normal cleavage products at the +1 position. The cleavage at the -8 position was essentially effective only in the presence of C5 protein. Possible secondary structures for cleavage at the -8 position deviate significantly from the structures of the known RNase P substrates, implying that C5 protein can allow the enzyme to broaden the substrate specificity more than previously appreciated.
대장균의 RNase P는 tRNA의 5’ 말단을 가공하는 효소로 효소 활성 요소인 M1 RNA와 단백질 보조인자인 C5 단백질로 구성되어 있다. RNA 결합 단백질로서의 C5 단백질의 기능을 이해하기 위해서 C5 단백질에 결합하는 RNA aptamer를 SELEX 기술을 이용하여 선별하고 그 특성을 고찰하였다. C5 단백질은 아주 다양한 RNA 구조와 대단히 강력하고 특이적으로 결합을 하였다. C5 단백질에 결합하는 RNA의 중심 구조는 중심부 고리가 대단히 큰 머리핀 구조로 결정되었다. 선별된 RNA는 M1 RNA에 비해서는 그 복합체 안정성이 떨어졌으나 일반적인 RNA-단백질 복합체에 비해서는 안정한 성질을 보였으며, 마그네슘 이온에 대한 결합의 의존성은 M1 RNA와 정반대의 경향을 보였다. 반면 암모늄 이온에 대한 결합 의존성은 M1 RNA와 비슷하게 소수성을 보였다. 실제 대장균 내에서 M1 RNA 외에 C5 단백질과 결합하는 다른 종류의 RNA가 존재하는지 알아보기 위해 genomic SELEX를 수행하였다. 역시 다양한 RNA들이 결합을 하였다. 특이하게 지금까지 알려지지 않았던 RNA들이 관찰되었는데 이중 ycaH 유전자에 상보적인 RNA가 C5 단백질에 결합하는 것으로 나타났다. 이 RNA가 실제 대장균 내에 존재하는지 알아본 결과 대장균 내에 성장 과정에 관계 없이 항상 존재하고 C5 단백질에 의존적으로 RNase P에 의해 절단되었다. 이런 단백질 의존적 RNase P 반응이 어떤 기작으로 일어나는지 알아보기 위해 돌연변이화 된 tRNA를 이용하여 동역학적 계수를 알아보았다. 이를 통해 C5 단백질이 RNase P 반응에 있어 기질의 특이성을 이전에 알려진 것보다 더 넓혀줄 수 있다는 것을 알 수 있었다.
