Swapping the domains of exoribonucleases RNase II and RNase R: Conferring upon RNase II the ability to degrade ds RNA

Abstract

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.

rnr Gene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

“…A lesser activity at 37°C and above, compared to 22°C, hinted that the enzyme might not be heat stable. To test this, RNase R Ps was preincubated at different temperatures (22,37,45,50, and 60°C) for 5 min, followed by assay for activity at 22°C. No activity was observed when the enzyme was preincubated at the temperatures above 37°C, suggesting that the RNase R Ps is extremely heat labile (Fig.…”

“…Interestingly, M. genitalium RNase R (RNase R Mg ) was shown to produce mostly trinucleotides (and only a small amount of dinucleotides) as the end products (19). Although the molecular basis of the production of different lengths of end products of RNA chain by different RNase R homologues is not known, the proposed catalytic pocket residues in the RNB domain of the enzyme might play an important role, as shown for RNase II and RNase R from E. coli (3,12,21,22). Structural models of RNase R Ec (3,34) based on the RNase II crystal structures (15,35) have placed the catalytic site deep in a putative funnel-like structure of RNB domain into which the 3Ј ends of the RNA chains are threaded for cleavage.…”

“…Therefore, it is possible that the strength of anchoring of RNA 3Ј ends in the active-site domain of RNase R of different organisms may vary, leading to the release of different end products which fail to remain bound to the enzyme. It is important to note that Pseudomonas species contain only one member of RNB/RNR exoribonuclease family (based on sequenced genomes), which has greater identity to RNase R. The possibility that RNase R Ps maintains certain features of RNase II, such as the generation of 4 nt as a limit product, possibly due to sequence conservation in the RNB site (22), was not obvious from a sequence alignment of RNase R Ps , RNase R Ec , and RNase II Ec (see Fig. S6 in the supplemental material).…”

The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection

“…E. coli RNase R has at the C terminus a region rich in lysines (Fig. 1A), which was shown to be involved in the degradation of double-stranded substrates, probably by helping to unwind the two strands (42). This region is absent in Cj-RNR.…”

Bacterialtrans-Translation: From Functions to Applications

“…In E. coli, RNase R is a 92 kDa ubiquitous protein encoded by the rnr gene, and is involved in the degradation of different types of structured RNAs such as rRNA, small RNA, and mRNA [79,80]. RNase R belongs to the RNase II superfamily of enzymes that degrade RNA hydrolytically in the 3 → 5 direction in a processive and sequence-independent manner.…”