Hannon G J, Maroney P A, Branch A, Benenfield B J, Robertson H D, Nilsen T W
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Mol Cell Biol. 1989 Oct;9(10):4422-31. doi: 10.1128/mcb.9.10.4422-4431.1989.
We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.
我们在此报告，人18S rRNA成熟的5'末端是通过两步加工反应在体外产生的。第一步，SP6转录本在HeLa细胞核仁提取物中于靠近外部转录间隔区（ETS）-18S边界的三个位置被特异性切割。在这些切割位点中，两个是主要的，另一个是次要的。RNase T1指纹图谱和二级核酸酶分析表明，两个主要切割位点位于18S rRNA成熟5'末端上游3个和8个碱基处，次要切割位点位于18S序列内1个碱基处。所有这三次切割均产生5'-羟基、2'-3'-环磷酸末端，且位于序列UACCU中腺苷残基的5'端，该序列在ETS-18S边界附近重复了三次。第二步，含有3个ETS碱基的初始切割产物通过HeLa细胞胞质提取物中的一种活性转化为5'末端与成熟18S RNA相同的RNA。
