engleski

Identification of novel regulation of Cas3 activity in Escherichia coli.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitutes an adaptive immune system in bacteria and archaea against invading DNA such as viruses and plasmids. CRISPR-Cas defence operates through targeting of RNA to complementary sequences in invader DNA or RNA, divided into three stages: adaptation, expression and maturation, and interference. During interference stage, a protein complex called Cascade forms an R-loop by base pairing crRNA to protospacer dsDNA, displacing the DNA strand that is not complementary to crRNA. Cas3 is then recruited to bind the R-loop and Cas3 nuclease initiates degradation of invader DNA by first introducing nicks at the exposed ssDNA of the R-loop, followed by extensive exonuclease activity in a 3’ to 5’ unidirectional manner. We observed that E. coli Type I-E CRISPR-Cas mediated immunity to phage lambda was responsive to the temperature of incubation and growth phase if cas genes were expressed from their native promoters in cells lacking H-NS repressor. Genetic analysis indicated that cas3 gene was a limiting factor for efficient resistance to phage infection. This was based on observations that despite increased transcription levels of cas3 gene in hns mutants at both temperatures in stationary cells, the immunity to phage lambda was abolished and dependent on expression of the HtpG chaperone (high-temperature protein G) at elevated temperature (37°C). Here I will present current studies of Cas3 protein and transcription levels, the role of HtpG and changes in secondary structure of purified Cas3 protein at different temperatures.

CRISPR-cas ; Cas3 ; HtpG, E. coli

nije evidentirano