The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8.

BACKGROUND: RNA metabolism, including RNA synthesis and RNA degradation, is one of the most conserved biological systems and has been intensively studied; however, the degradation network of ribonucleases (RNases) and RNA substrates is not fully understood. RESULTS: The genome of the extreme thermophile, Thermus thermophilus HB8 includes 15 genes that encode RNases or putative RNases. Using DNA microarray analyses, we examined the effects of disruption of each RNase on mRNA abundance. Disruption of the genes encoding RNase J, RecJ-like protein and RNase P could not be isolated, indicating that these RNases are essential for cell viability. Disruption of the TTHA0252 gene, which was not previously considered to be involved in mRNA degradation, affected mRNA abundance, as did disruption of the putative RNases, YbeY and PhoH-like proteins, suggesting that they have RNase activity. The effects on mRNA abundance of disruption of several RNase genes were dependent on the phase of cell growth. Disruption of the RNase Y and RNase HII genes affected mRNA levels only during the log phase, whereas disruption of the PhoH-like gene affected mRNA levels only during the stationary phase. Moreover, disruption of the RNase R and PNPase genes had a greater impact on mRNA abundance during the stationary phase than the log phase, whereas the opposite was true for the TTHA0252 gene disruptant. Similar changes in mRNA levels were observed after disruption of YbeY or PhoH-like genes. The changes in mRNA levels in the bacterial Argonaute disruptant were similar to those in the RNase HI and RNase HII gene disruptants, suggesting that bacterial Argonaute is a functional homolog of RNase H. CONCLUSION: This study suggests that T. thermophilus HB8 has 13 functional RNases and that each RNase has a different function in the cell. The putative RNases, TTHA0252, YbeY and PhoH-like proteins, are suggested to have RNase activity and to be involved in mRNA degradation. In addition, PhoH-like and YbeY proteins may act cooperatively in the stationary phase. This study also suggests that endo-RNases function mainly during the log phase, whereas exo-RNases function mainly during the stationary phase. RNase HI and RNase HII may have similar substrate selectivity.

An alkyltransferase-like protein from Thermus thermophilus HB8 affects the regulation of gene expression in alkylation response.

Alkylation is a type of stress that is fatal to cells. However, cells have various responses to alkylation. Alkyltransferase-like (ATL) protein is a novel protein involved in the repair of alkylated DNA; however, its repair mechanism at the molecular level is unclear. DNA microarray analysis revealed that the upregulation of 71 genes because of treatment with an alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine was related to the presence of TTHA1564, the ATL protein from Thermus thermophilus HB8. Affinity chromatography showed a direct interaction of purified TTHA1564 with purified RNA polymerase holoenzyme. The amino acid sequence of TTHA1564 is homologous to that of the C-terminal domain of Ada protein, which acts as a transcriptional activator. These results suggest that TTHA1564 might act as a transcriptional regulator. The results of DNA microarray analysis also implied that the alkylating agent induced oxidation stress in addition to alkylation stress.