Sirum-Connolly K, Peltier J M, Crain P F, McCloskey J A, Mason T L
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003-4505, USA.
Biochimie. 1995;77(1-2):30-9. doi: 10.1016/0300-9084(96)88101-6.
The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.
核糖体大亚基RNA肽基转移酶区域的序列和结构高度保守，特定的修饰核苷酸可能是负责肽键形成的催化中心中重要的结构或功能元件。事实上，从23S rRNA的体外转录本和完整的50S蛋白中无法重构有活性的大肠杆菌50S亚基。因此，酵母的PET56基因编码一种必需的核糖甲基转移酶，该酶特异性修饰线粒体大核糖体RNA（21S）肽基转移酶中心一个普遍保守的核苷酸G2270，这一点具有重要意义。由于酵母线粒体21S rRNA中这种修饰的缺失严重影响54S亚基的组装，大肠杆菌23S rRNA中2251位类似的2'-O-甲基鸟苷（Gm2251）可能也是50S亚基组装所必需的。Gm可能是rRNA正确折叠、一种或多种核糖体蛋白结合或rRNA与tRNA相互作用的关键结构决定因素。先前的研究表明，相对于大肠杆菌和真核细胞质rRNA，线粒体大rRNA的修饰程度最低。通过使用高效液相色谱-质谱联用的直接化学分析方法，重新检测了酵母线粒体rRNAs的修饰状态，发现21S rRNA中存在Gm、Um和假尿苷（ψ）。Um定位于核苷酸2791，它对应于大肠杆菌23S rRNA中核糖甲基化且普遍保守的U2552，而ψ最近定位于2819位，它对应于大肠杆菌23S rRNA中的ψ2580。在其他修饰程度最低的线粒体rRNAs的肽基转移酶中心保留Um和ψ核苷酸，表明这些修饰可能像Gm2270一样，对核糖体组装或功能或两者都至关重要。
