Functional analysis of a codon-independent release factor YaeJ

YaeJ蛋白是一种不依赖密码子的释放因子，具有肽基-tRNA水解酶(PTH)活性，在大肠杆菌中作为停滞核糖体救援因子发挥作用。为了确定YaeJ功能所需的残基，我们对体外PTH活性进行了突变分析，以挽救停滞在不间断mRNA上的核糖体，并对核糖体结合效率进行了分析。我们重点研究了细菌YaeJ蛋白之间的保守残基。虽然在结构化的GGQ结构域中没有对PTH活性重要的YaeJ特异性残基，但PTH活性需要在随后的C末端延伸中的Arg118、Leu119、Lys122、Lys129和Arg132中进行。所有这些残基在细菌中都是完全保守的。这些残基中每个残基的单一氨基酸替换显著降低了核糖体结合效率。这些生化发现为了解YaeJ如何进入停滞的核糖体的A位提供了线索。

The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a nonstop mRNA, and for ribosome binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129, and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. Single amino acid substitutions for each of these residues significantly decreased ribosome binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.