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...
