Bocchetta M, Xiong L, Mankin A S
Center for Pharmaceutical Biotechnology-m/c 870, University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3525-30. doi: 10.1073/pnas.95.7.3525.
rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.
核糖体RNA（rRNA）在肽基转移酶的功能中发挥着重要作用，肽基转移酶是核糖体的催化中心，负责肽键的形成。肽基转移酶的底物，即肽基-tRNA和氨酰-tRNA的正确定位，对于转肽反应的催化和蛋白质合成至关重要。在本报告中，我们确定了一小部分rRNA核苷酸，它们最有可能直接参与在大核糖体亚基功能位点上tRNA的结合。通过将生物素化的tRNA底物与来自大肠杆菌的随机修饰的大核糖体亚基结合，并在抗生物素蛋白树脂上捕获形成的复合物，我们在大核糖体亚基rRNA中鉴定出四个核苷酸（位置G2252、A2451、U2506和U2585），其修饰会阻止肽基-tRNA类似物在P位点的结合，以及一个残基（U2555），其修饰会干扰肽基部分向嘌呤霉素的转移。这些核苷酸代表了受tRNA类似物保护免受化学修饰的位置的一个子集，并显著缩小了可能直接参与核糖体功能位点上tRNA结合的23S rRNA核苷酸的数量。
