Mohan A, Whyte S, Wang X, Nashimoto M, Levinger L
Department of Natural Sciences/Biology, York College, City University of New York, Jamaica 11451, USA.
RNA. 1999 Feb;5(2):245-56. doi: 10.1017/s1355838299981256.
Cytoplasmic tRNAs undergo posttranscriptional 5' and 3' end processing in the eukaryotic nucleus, and CCA (which forms the mature 3' end of all tRNAs) must be added by tRNA nucleotidyl transferase before tRNA can be aminoacylated and utilized in translation. Eukaryotic 3'-tRNase can endonucleolytically remove a 3' end trailer by cleaving on the 3' side of the discriminator base (the unpaired nucleotide 3' of the last base pair of the acceptor stem). This reaction proceeds despite a wide range in length and sequence of the 3' end trailer, except that mature tRNA containing the 3' terminal CCA is not a substrate for mouse 3'-tRNase (Nashimoto, 1997, Nucleic Acids Res 25:1148-1154). Herein, we extend this result with Drosophila and pig 3'-tRNase, using Drosophila melanogaster tRNAHis as substrate. Mature tRNA is thus prevented from recycling through 3' end processing. We also tested a series of tRNAs ending at the discriminator base (-), with one C added (+C), two Cs added (+CC), and CCA added (+CCA) as 3'-tRNase inhibitors. Inhibition was competitive with both Drosophila and pig 3'-tRNase. The product of the 3'-tRNase reaction (-) is a good 3'-tRNase inhibitor, with a KI approximately two times KM for the normal 3'-tRNase substrate. KI increases with each nucleotide added beyond the discriminator base, until when tRNA+CCA is used as inhibitor, KI is approximately forty times the substrate KM. The 3'-tRNase can thus remain free to process precursors with 3' end trailers because it is barely inhibited by tRNA+CCA, ensuring that tRNA can progress to aminoacylation. The active site of 3'-tRNase may have evolved to make an especially poor fit with tRNA+CCA.
细胞质转运RNA（tRNA）在真核细胞核中经历转录后5'和3'末端加工，并且在tRNA能够被氨酰化并用于翻译之前，必须由tRNA核苷酸转移酶添加CCA（其形成所有tRNA的成熟3'末端）。真核3'-tRNase可以通过在鉴别碱基（受体茎最后一个碱基对的3'端未配对核苷酸）的3'侧切割来内切核酸酶去除3'末端拖尾序列。尽管3'末端拖尾序列的长度和序列范围很广，但该反应仍会进行，除了含有3'末端CCA的成熟tRNA不是小鼠3'-tRNase的底物（Nashimoto，1997，《核酸研究》25:1148 - 1154）。在此，我们以果蝇黑腹果蝇tRNAHis为底物，用果蝇和猪的3'-tRNase扩展了这一结果。因此，成熟tRNA被阻止通过3'末端加工进行循环利用。我们还测试了一系列在鉴别碱基处结束的tRNA（-），分别添加一个C（+C）、两个C（+CC）和CCA（+CCA）作为3'-tRNase抑制剂。抑制作用对果蝇和猪的3'-tRNase均具有竞争性。3'-tRNase反应的产物（-）是一种良好的3'-tRNase抑制剂，其抑制常数（KI）约为正常3'-tRNase底物米氏常数（KM）的两倍。随着在鉴别碱基之外添加的每个核苷酸，KI都会增加，直到当tRNA + CCA用作抑制剂时，KI约为底物KM的40倍。因此，3'-tRNase可以自由地处理带有3'末端拖尾序列的前体，因为它几乎不受tRNA + CCA的抑制，确保tRNA能够进行氨酰化。3'-tRNase的活性位点可能已经进化到与tRNA + CCA的契合度特别差。
