Nashimoto M, Tamura M, Kaspar R L
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA.
J Mol Biol. 1999 Apr 9;287(4):727-40. doi: 10.1006/jmbi.1999.2639.
Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.
哺乳动物tRNA 3'加工内切核糖核酸酶（3' tRNase）通过在鉴别核苷酸下游紧邻位置切割RNA，从前体tRNA中去除3'拖尾序列。尽管3' tRNase能够识别并切割任何与另一种RNA形成类似前体tRNA复合物的靶RNA，但在某些情况下，切割会发生在鉴别核苷酸附近的多个位点。我们使用各种前体tRNAArg变体和纯化的猪酶，研究了前体tRNA的哪些特征决定切割位点。由于T茎环以及受体茎加上3'拖尾序列足以被3' tRNase识别，我们构建了在T茎和/或受体茎中存在碱基对添加和/或缺失的变体。缺少一个和两个受体茎碱基对的前体tRNA分别在鉴别核苷酸下游一个和两个核苷酸以及两个和三个核苷酸处被切割。另一方面，含有额外受体茎碱基对的前体tRNA变体仅在鉴别核苷酸之后被切割。通过从T茎中删除一个碱基对，切割位点向鉴别核苷酸下游一个和两个核苷酸处移动，但T茎中额外的碱基对并未改变切割位点。包含八个碱基对受体茎加上六个碱基对T茎、八个碱基对受体茎加上四个碱基对T茎或六个碱基对受体茎加上六个碱基对T茎的前体tRNA变体均在原始核苷酸之后被切割。一般来说，受体茎和T茎中总共超过11个碱基对的前体tRNA变体仅在鉴别核苷酸之后被切割，而总共具有N个碱基对（N小于12）的前体tRNA变体在鉴别核苷酸下游12 - N和13 - N个核苷酸处被切割。变体的切割效率根据与真实前体tRNA的结构变化程度而降低。这表明受体茎和T茎的碱基对数对于3' tRNase的识别和切割都很重要。
