Structural And Functional Studies On The Key Ribonuclease Of The Gram-positive Bacterial RNA Degradosome

The RNA degradosome is an important enzyme complex involved in rRNA maturation and mRNA degradation,which consists of nucleases,RNA helicase and glycolytic enzymes.Among these enzymes,RNase E,RNase Y and RNase J are the key ribonucleases.While RNase E is involved in RNA degradation in Gram-negative bacteria such as Escherichia coli.RNase Y and RNase J play a crucial role in RNA processing in Gram-positive bacteria.And it has been proposed that RNase Y is a isozyme of RNase E,which functions as a clamp protein.Deletion of RNase Y affected the growth rate of Gram positive bacteria as well as the cell morphology,sporulation and antibiotic sensitivity,which is also related to the regulation of toxic gene expression in cells.In addition,RNA degradation in Gram-positive bacteria requires RNase J,which contains both endonuclease and 5'-3'exonuclease activities.Despite that RNase J is essential for the growth of Streptococcus pyogenes,it can be completely knocked out in Bacillus subtilis and Staphylococcus aureus.The detailed mechanism of RNA degradation in Gram-positive bacteria is still unclear.Here,we reported the high-resolution crystal structure of the Deinococcus radiodurans RNase J(DraRNase J)in complexed with UMP and its catalytically inactive mutant complexed with 6 nt RNA in a functional form and carried out the biochemical experimental validation.The structures indicate that RNase J belongs to the ?-CASP subfamily of proteins and uses zinc ions for two-metal-ion catalysis.One residue conserved among RNase J orthologues forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization.A conserved 5'-monophosphate binding pocket was observed in RNase J-RNA structure,which determines the cleavage polarity of the DraRNase J.The additional manganese ion,which is coordinated by conserved residues at the dimer interface,is critical for DraRNase J dimerization and exo-and endonucleolytic activity switch.In addition,full-length rnj gene and the unconserved C terminus are both essential for cell growth,and that heterozygous mutants exhibited reduced growth rate and environmental stress adaptation.RNase Y interacts with a variety of RNA degradation-associated components including RNase J.We generated a homozygous knockedout strain of RNase Y in D.radiodurans.The mutant could further greatly knockdown the copy of rnj gene of D.radiodurans,resulting in a severely reduction of the growth rate of D.radiodurans and significantly affecting its extreme resistance.RNA-seq transcriptome sequencing analysis indicated that the absence of RNase Y led to the enrichment of transcripts involved in metabolism-related pathways,ion transport-related pathways,and proteins involved in DNA damage response,leading to the increase of genom instability of D.radiodurans,further leading to reduced genome stability in D.radiodurans.In addition,a series of potential RNase Y related operons were identified by comparing the transcriptome data between wild-type and mutant strains.