Substrate Binding and Active Site Residues in RNases E and G

Abstract

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5-sensor, the S1 domain, and the Mg ؉2 /Mn ؉2 binding sites. The RNase E/G family of bacterial endoribonucleases is widely distributed among bacteria (1). Both RNase E and RNase G are expressed in Escherichia coli. RNase E was first characterized as an essential processing enzyme required for the maturation of 5 S rRNA 2 (2, 3). It is now known also to be involved in processing the 5Ј-spacer region of 16 S rRNA (4), most tRNA precursors (5, 6), transfer messenger RNA (7), and in the metabolism of many small regulatory RNAs (8, 9). It is also responsible for catalyzing the initial cleavage in the degradation of most mRNAs (10, 11). Furthermore, RNase E is part of a larger complex, the RNA degradosome (12-14). In contrast, RNase G appears to play a more limited role in RNA metabolism. It is responsible for the formation of the mature 5Ј terminus of 16 S rRNA (4, 15) and participates in the degradation of a limited set of mRNAs (16,17). It is not essential, however. Although both enzymes prefer single-stranded substrates, neither displays stringent sequence specificity (18 -20). However, both enzymes are 5Ј-end-dependent; i.e. their activity is stimulated, both in vivo and in vitro by a 5Ј-monophosphorylated terminus on their substrates (21-26). To explain this observation, it was postulated that a 5Ј-phosphate binding pocket exists on the surface of these enzymes (24). This idea has been substantially verified by the crystal structure of the catalytic domain of RNase E in complex with a substrate analog (27). These authors showed that RNase E contains a 5Ј-sensor domain that can interact specifically with a 5Ј-monophosphorylated substrate via contacts with Gly-124, Val-128, Arg-169, and Thr-170 (27).Several investigations have identified potential RNA binding surfaces on RNase E in addition to the 5Ј-sensor, including an arginine-rich region (28 -30) and the S1 domain (31, 32). In addition, the active (catalytic) site itself must contribute to substrate binding. The arginine-rich region, however, lies outside the minimal N-terminal domain of RNase E that is sufficient for enzymatic activity (28 -30). Several residues in the S1 domain could contribute to RNA binding, but only three, Phe-57, Phe-67, and Lys-112 provide obvious contacts to the substrate (27). Thus, it is not clear to what extent the 5Ј-sensor contributes to substrate binding. Indeed, it has been suggested that interaction of RNase E or G with a 5Ј-monophosphorylated substrate increases these enzymes' V max , effectively providing activation of these enzymes (25). Because a crystal structure was not available at the time this work was initiated, we examined instead the role of two types of conserved amino acid residue lying between the S1 domain and residue 400 in RNa...

5′ End-independent RNase J1 Endonuclease Cleavage of Bacillus subtilis Model RNA

“…This is reminiscent of recent data from the Mackie lab on the 5Ј-phosphate sensor domain of RNase E (28,29). Mutation of a key residue in the domain of RNase E that is required in vitro for binding to a 5Ј-monophosphate group had relatively little effect on growth in vivo.…”

Small RNA-induced mRNA degradation achieved through both translation block and activated cleavage

“…Indeed, when RyhB pairs with a target mRNA and blocks translation initiation, the last translating ribosome can reach and pass the cleavage site before the degradosome initiates mRNA cleavage. Once the mRNA is cleaved, the resulting generation of a 59-P (monophosphorylated) end accelerates by a 30-fold factor the RNase E activity toward the 39 end of the transcript (Mackie 1998;Jiang and Belasco 2004;Garrey et al 2009). Consequently, the cleaved mRNA will be fully degraded more rapidly.…”

Altering the Divalent Metal Ion Preference of RNase E

“…Mutations were introduced into plasmids containing the catalytic domain of RNase E (residues 1 to 529) in the pET24b backbone using QuikChange mutagenesis as described previously (15,16). After the induction of the appropriate cultures, the wild-type, D303C, and D346C RNase E 1-529 enzymes were prepared as described previously (16).…”

“…No attempt was made to remove incomplete or truncated transcripts. Appropriate mutations were introduced into full-length rne ϩ constructs using the two step strategy outlined in Garrey et al (15,16). QuikChange mutagenesis was performed on a derivative of pUC19 containing an ϳ2.7-kb HindIII-SalI fragment encompassing amino acid residues 184 to 1061 plus the 3= untranslated region (UTR; "fragment 2" in reference 15).…”