Niu L, Lane B G, Ofengand J
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Biochemistry. 1999 Jan 12;38(2):629-35. doi: 10.1021/bi9821869.
A Bacillus subtilis ORF, ypul, 41% homologous to rsuA, the gene for the synthase which forms pseudouridine 516 in Escherichia coli 16S rRNA, was cloned and the protein expressed and affinity-purified by the His tag procedure. Reactions with E. coli 16S and 23S rRNA transcripts were performed in vitro. The protein did not form pseudouridine 516 as expected but did produce pseudouridine 552 in 16S rRNA and pseudouridines 1199, 2605, and 2833 in 23S rRNA. Of these, only pseudouridine 2605 is found naturally in either E. coli or B. subtilis rRNA. Kinetic experiments confirmed that pseudouridine 2605 was the primary target. Comparison of the four pseudouridine sites yielded a consensus recognition sequence for the synthase. This consensus sequence was not present at any other site in either E. coli or B. subtilis 16S or 23S RNA. We propose that YpuL is the B. subtilis pseudouridine 2633 (2605 in E. coli) synthase. Since the closest gene sequence homologue in E. coli is yciL, we suggest that its gene product is the corresponding E. coli pseudouridine 2605 synthase.
克隆了一个与rsuA有41%同源性的枯草芽孢杆菌开放阅读框ypul，rsuA是在大肠杆菌16S rRNA中形成假尿苷516的合酶基因，通过His标签程序表达并亲和纯化了该蛋白。体外进行了与大肠杆菌16S和23S rRNA转录本的反应。该蛋白并未如预期形成假尿苷516，但在16S rRNA中产生了假尿苷552，在23S rRNA中产生了假尿苷1199、2605和2833。其中，只有假尿苷2605在大肠杆菌或枯草芽孢杆菌rRNA中天然存在。动力学实验证实假尿苷2605是主要靶点。对这四个假尿苷位点的比较产生了该合酶的共有识别序列。该共有序列在大肠杆菌或枯草芽孢杆菌16S或23S RNA的任何其他位点均不存在。我们提出YpuL是枯草芽孢杆菌假尿苷2633（大肠杆菌中为2605）合酶。由于大肠杆菌中最接近的基因序列同源物是yciL，我们认为其基因产物是相应的大肠杆菌假尿苷2605合酶。
