Stade K, Jünke N, Brimacombe R
Max-Planck-Institut für Molekulare Genetik (AG Ribosomen, Berlin (Dahlem), Germany.
Nucleic Acids Res. 1995 Jul 11;23(13):2371-80. doi: 10.1093/nar/23.13.2371.
Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes. The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome. Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures. The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids). Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781. Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I. These cross-linking results are correlated with other types of topographical data relating to the 50S subunit.
由合适的mRNA片段编码的不同长度的肽在大肠杆菌核糖体上原位生物合成。这些肽在其N端甲硫氨酸残基上连接有一个重氮丙啶衍生物，当肽仍附着在核糖体上时，该衍生物会被光激活。随后，通过我们的标准程序分析与23S RNA发生光交联的位点。长度不断增加的肽的N端逐渐与核苷酸750（6、9或13 - 15个氨基酸的肽）、核苷酸1614交联，同时与核苷酸1305和1350之间的第二个位点（25 - 26个氨基酸的肽）以及核苷酸91（29 - 33个氨基酸的肽）交联。此前我们已经表明，1或2个氨基酸的肽与肽基转移酶环内的核苷酸2062、2506和2585交联，而三肽和四肽还与核苷酸2609和1781交联。综合来看，这些数据表明新生肽链的路径从23S RNA结构域V中的肽基转移酶环移动到结构域IV，然后到结构域II，再到结构域III，最后到结构域I。这些交联结果与其他与50S亚基相关的拓扑学数据相关。
