engleski

The role of interaction between HtoG and Cas3 proteins on activity of the Escherichia coli Type I-E CRISPR-Cas system

CRISPR (clustered regularly interspaced short palindromic repeats) and its associated Cas proteins provides many bacteria and archaea with a defence mechanism by RNA mediated targeting of invading genetic elements. Escherichia coli type I-E CRISPR-Cas system detects invading DNA by a "Cascade" nucleoprotein surveillance complex that contains CRISPR RNA (crRNA) bound within that recognizes target sites on invader DNA. This triggers R-loop formation and binding of Cas3 helicase-nuclease that degrades invader DNA. Previous studies in E. coli showed that CRISPR-Cas activity also depends on the activity of the high-temperature protein G (HtpG) which is important for maintaining functional levels of Cas3, and the transcriptional regulator H-NS which represses Cascade and crRNA expression. Factors that control transcription of E. coli cas3 gene have not been identified. We have recently found that H-NS repressor also represses expression of the cas3 gene. Transcription of the cas3 gene was elevated in cells lacking H-NS if grown to stationary phase, but surprisingly the activity of the Cas3 protein was strongly dependent on increased expression of either Cas3 or HtpG chaperone from plasmid only at 37°C but not at 30°C. The precise role of HtpG in E. coli is unknown and the temperature-sensitive phenotype of Cas3 or HtpG was not reported before. In addition, interaction between HtpG and Cas3 is not well understood and was not investigated in vitro. We would like to the test if the molecular interactions between purified Cas3 and HtpG proteins (each labelled with 6xHis tag at N-terminus) are different if proteins are incubated or extracted from cells at different temperatures of incubation.

CRISPR-Cas; H-NS; Cas3; HtpG; E. coli

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