Rao Arasada Rajeswara, Varshney Umesh
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Microbiology (Reading). 2002 Dec;148(Pt 12):3913-20. doi: 10.1099/00221287-148-12-3913.
Ribosome recycling factor (RRF), coded for by the frr locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles. In a cross-species-complementation experiment, it was shown that the Thermus thermophilus RRF protein lacking five amino acids from its C-terminal end (deltaC5TthRRF) but not the full-length protein (TthRRF) complemented Escherichia coli for its frr(ts) phenotype. It was also shown that the Mycobacterium tuberculosis RFF protein (MtuRRF) did not complement E. coli LJ14 for frr(ts). However, simultaneous expression of elongation factor G (EFG) and RRF from M. tuberculosis resulted in complementation of E. coli LJ14. Here it is shown that unlike deltaC5TthRRF, an equivalent mutant of MtuRRF lacking six amino acids from its C-terminal end (deltaC6MtuRRF) did not complement E. coli LJ14. Surprisingly, deltaC6MtuRRF failed to complement the strain even in the presence of homologous EFG (MtuEFG). The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly. However, ribosome-binding assays showed that the mutant protein was compromised in its binding to E. coli ribosomes. It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.
核糖体循环因子（RRF）由frr基因座编码，参与细菌和真核细胞器中终止后复合物的拆解以及核糖体的循环利用，以便进行新一轮的起始过程。在一项跨物种互补实验中，结果表明，嗜热栖热菌的RRF蛋白（TthRRF）C末端缺失五个氨基酸的突变体（deltaC5TthRRF）而非全长蛋白（TthRRF）能够互补大肠杆菌的frr(ts)表型。实验还表明，结核分枝杆菌的RRF蛋白（MtuRRF）不能互补大肠杆菌LJ14的frr(ts)表型。然而，同时表达结核分枝杆菌的延伸因子G（EFG）和RRF可使大肠杆菌LJ14得到互补。本文表明，与deltaC5TthRRF不同，MtuRRF的一个等效突变体，即C末端缺失六个氨基酸的突变体（deltaC6MtuRRF）不能互补大肠杆菌LJ14。令人惊讶的是，即使存在同源的EFG（MtuEFG），deltaC6MtuRRF仍无法互补该菌株。这些蛋白的生化和生物物理特性表明，突变的RRF能够正确折叠。然而，核糖体结合试验表明，突变蛋白与大肠杆菌核糖体的结合能力受损。研究表明，RRFs C末端的保守氨基酸有助于它们在核糖体上的停留，并且RRF与EFG之间的特定相互作用在终止复合物的拆解过程中至关重要。
