(The) effects of escherichia coli SecA binding to RNA on the translation of secARNA에 대한 대장균 SecA의 결합이 SecA 유전자의 해독에 미치는 영향

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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 -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 -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.
Kim, Hyoung-Man김형만
한국과학기술원 : 생물과학과,
Issue Date
135368/325007 / 000963401

학위논문(석사) - 한국과학기술원 : 생물과학과, 1998.2, [ vi, 41 p. ]


해독; Translation; Galactosidase; 돌연변이; Mutation; SecA

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