YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity

Abstract

Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E. coli and its human homolog Sfn in a screen for proteins that are regulated by 3′-phosphoadenosine 5′-phosphate (pAp). Here, we identify YtqI as a potential functional analog of Orn through its interaction with pAp. YtqI degrades RNA oligonucleotides in vitro with preference for 3-mers. In addition, YtqI has pAp-phosphatase activity in vitro. In agreement with these data, YtqI is able to complement both orn and cysQ mutants in E. coli. An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli. Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

“…In the case of NrnA, having considered the possibility that 3-mers might be a preferred substrate of this enzyme, we indeed found turnover numbers to be 10-fold higher for 3-mers (59Cy5-CCC-39) than for 5-mers (59Cy5-CCCCC-39; 1.5 vs. 0.14 pmol/mg/min) (Mechold et al 2007). We therefore decided to compare turnover of 5-mers and 2-mers when degraded by Rv2837c.…”

“…According to previous observations with NrnA (Mechold et al 2007), we expected its homologs to degrade nanoRNA in the 39-59 direction. We therefore used nanoRNA substrates that were 59-labeled with Cy5 as they should allow intermediate products of degradation to be detected.…”

“…This mutant (strain UM341, see Table 1) (Mechold et al 2007) uses the anhydrotetracycline (Atc)-inducible promoter P LtetO-1 (Lutz and Bujard 1997) together with a Tet-repressor (TetR) to ensure tight control in the absence of Atc. A growth defect of UM341 could easily be observed in the absence of Atc; transformants of this strain with pBAD18 produced pinpoint-sized colonies that stopped growing, whereas transformants with the orncarrying plasmid (pUM408) were significantly larger after overnight growth and continued to grow (Fig.…”

“…This constraint has therefore to be considered in the design of synthetic cells. We previously introduced the term ''nanoRNA'' in order to distinguish these extremely short oligonucleotides of five residues and shorter in length from the longer microRNAs with known explicit functions in the cell (Mechold et al 2007;Fang et al 2009). …”

“…We demonstrated in a previous work that in B. subtilis, the situation is quite different from that in E. coli; here, the degradation of nanoRNA is catalyzed by multiple redundant RNases (Fang et al 2009). We have identified two enzymes with nanoRNase activity in B. subtilis: YtqI (now renamed NrnA for nanoRNase A) (Mechold et al 2007) and YngD (now NrnB) (Fang et al 2009). Remarkably, NrnA was shown not only to be required for scavenging nanoRNAs but also to be necessary for the dephosphorylation of 39-phosphoadenosine 59-phosphate (pAp), a side-product of sulfur assimilation and acyl carrier proteins (ACPs) synthesis.…”