SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its translation during normal protein secretion by binding to a secretion-responsive element located near the 5end of its gene on geneX-secA mRNA. It was reported that the overexpression of the SecA protein severely reduced only the expression of secA-lacZ fusion. The RNA binding domain of SecA protein, whose binding to its mRNA represses the translation of the mRNA, was investigated using a combination of genetic and biochemical approaches. Recently, SecA was found to have a sequence homology with superfamily II helicase having 6 conserved motifs. Subsequently, the helicase activity of SecA was confirmed. In order to investigate RNA binding property of SecA, the most conserved five amino acids of the 6th motif, the RNA binding motif, were substituted by alanine using an oligonucleotide-directed mutagenesis. The β-galactosidase activity of MM171.3 of the Escherichia coli strain with secA-lacZ translational fusion on its chromosomal DNA and the plasmid having base substitutions was measured. It was shown that the translation efficiency was derepressed in two mutants of the five mutants. Furthermore, the binding of mutant SecA proteins to geneX-secA intergenic RNA was studied using nitrocellulose filter binding assay. This analysis showed that the two mutants with high translation efficiency have lower binding affinity for geneX-secA intergenic RNA than the other proteins. This is in good agreement with the β-galactosidase activity assay. These results suggest strongly that the RNA binding domain of SecA plays an important role in the regulation of secA expression strengthening the earlier observation of helicase activity of SecA.
SecA 단백질은 대장균에서 단백질의 이동에 관여하는 ATPase이다. GeneX-secA의 전사 RNA의 유전자 사이 부분에 SecA 단백질이 붙어 자기 가신의 발현을 억제하는 자가 조절 기작으로 SecA 단백질의 발현이 조절된다. 유전자학적, 생화학적 접근방법으로 통해 SecA 단백질이 자신의 전사 RNA에 붙은 부분을 규명했다. 최근, SecA 단백질이 helicase 활성도가 있음이 보고되었으며 superfamily Ⅱ에 속하는 helicase임이 아미노산 서열 분석 결과 제시되었다. Helicase는 6개의 보존서열을 가지는데 이중 6번재 부분은 RNA 붙는 부분이라고 생각된다. 이 부분에 아미노산을 Alanine으로 바꾸는 돌연변이체를 만들어 해독 효율과 RNA 와의 해리상수를 구하고 이를 바탕으로 어느 아미노산이 RNA 결합에 중요한지 규명했다. 5개의 돌연변이체중 아미노산 서열중 573번과 577번이 자가 조절을 위해 RNA에 결합하는데 중요한 아미노산이라는 것을 말해준다.
