Study On The Enzyme Activity Of RibonucleaseⅢ Of Brucella Melitensis

Brucella is a genus of Gram-negative, intracellular bacteria that establishes chronic infection after invading into human and animal cells, but the mechanism how it avoids the host immune system is not well known. Recent evidences showed that Ribonucleaselll(RNaselll), RNaseE and Hfq can cooperatively regulate non-coding RNA expression, further affect the expression of other genes. RNaselll is a highly conserved enzyme family, widely exists in bacteria, fungus and eukaryocyte. All perfect paired RNAs which can form double strand structure can be cleaved by RNaselll, including bacterial RNAs and virus RNAs. Brucella modulates secretory trafficking via multiple Type4secretion effector proteins. Whether RNaseⅢ of Brucella can be secreted into host cells via Type4secretion system, affecting host gene expression on posttranscriptional level has not been reported.In this study, rncS gene of strain Brucella melitensis, which encodes RNaseⅢ, and its mutants rncS(E54A,D61A,E81A,E133A) were cloned by polymerase chain reaction and direct mutation. BM-pri-0015and BM-pre-0015contain base-paired structure predicted by RNASTRUCTURE4.6, pre-miRNAs Hsa-let-7a-1, Hsa-mir-16-1, PRV-LLT1and PRV-LLT2obtained from miRBase were selected to be the substrates of RNaselll. Firstly, the biochemical cleavage activity of RNaselll on different substrates was analysed: including the effect of Mg2+, Mn2+, Na+, K+and pH on RNaselll cleavage activity. Secondly, the cleavage activity of RNaselll (mutant) on BM-pre-0015was analysed. The results are as follows:The biochemical properties:1. Different RNA substrates can bind with RNaselll according to the EMSA experiment, showing no sequence specificity.2. Mg2+or Mn2+is essential for the cleavage activity of RNaselll according to the cleavage efficiency on different substrates, although the best concentration of metal ion for cleavage is different based on different substrates.3. Na+or K+is not essential for the cleavage activity of RNaselll according to the cleavage activity on different substrates.4. The cleavage activity of RNaseⅢ is higher in the condition of pH>8.0, otherwise is very low.5. The133th amino acid is essential for the cleavage activity of RNaselll.The substrates of RNaselll has no sequence specific accoding to the biochemistry analysis in this project. The best reaction condition and essential amino acid was validated, providing the necessary enzyme for the study of non-coding RNA.