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...

SummaryViable mutations affecting the 5Ј-phosphate sensor of RNase E, including R169Q or T170A, become lethal when combined with deletions removing part of the non-catalytic C-terminal domain of RNase E. The phosphate sensor is required for efficient autoregulation of RNase E synthesis as RNase E R169Q is strongly overexpressed with accumulation of proteolytic fragments. In addition, mutation of the phosphate sensor stabilizes the rpsT P1 mRNA as much as sixfold and slows the maturation of 16S rRNA. In contrast, the decay of other model mRNAs and the processing of several tRNA precursors are unaffected by mutations in the phosphate sensor. Our data point to the existence of overlapping mechanisms of substrate recognition by RNase E, which lead to a hierarchy of efficiencies with which its RNA targets are attacked.

Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudometallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5 ′ -exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.

The highly conserved branch point sequence (BPS) of UAC-UAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA (30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.The branch point-binding protein (BBP) 4 in Saccharomyces cerevisiae is an RNA-binding protein that is thought to be the first protein to bind the branch point sequence (BPS) during splicing. In yeast BBP binds to the highly invariant branch point sequence UACUAAC (branch point A is underlined), recognizing every nucleotide (1). BBP binds the BPS in the second commitment complex (CC2) and is necessary for stable formation of this complex (2). Splicing factor 1 (SF1), the human orthologue of BBP, is a more promiscuous RNA-binding protein, recognizing a degenerate sequence, CURAY (R ϭ purine, Y ϭ pyrimidine), in which only the U and A are critical (1). When discussing the yeast branch point-binding protein we will use the term BBP, when discussing the human protein we will use the term SF1, and when discussing both proteins we will use the term BBP/SF1. In the mammalian system, the equivalent complex to the yeast CC2 is the E complex in which SF1 binds the BPS (3, 4). In addition to recognizing the BPS, BBP/SF1 plays an important role in bridging the 5Ј end and 3Ј end of the intron by interacting with U1 snRNP at the 5Ј splice site and Mud2p at the 3Ј end of yeast introns and U2AF65 at the 3Ј end of human introns (5-8). BBP/SF1 is replaced at the BPS by U2 snRNP in an ATP-dependent step that leads to the formation of A complex (9, 10).BBP/SF1 contains multiple domains, many of which are conserved between yeast and human (Fig. 1). The BBP/SF1 N-terminal domain interacts with Mud2p in yeast and U2AF65 in humans (5,6,8). The C terminus contains a proline-rich domain, which in yeast interacts with Prp40, a component of the U1 snRNP (7). FBP11, a potential vertebrate homologue of Prp40, binds to the proline-rich domain of human SF1 (11).One of the most highly conserved regions of BBP/SF1 encompasses the RNA binding domains (Fig. 1). The region of BBP/SF1 responsible...

In yeast (Saccharoymces cerevisiae), the branchpoint binding protein (BBP) recognizes the conserved yeast branchpoint sequence (UACUAAC) with a high level of specificity and affinity, while the human branchpoint binding protein (SF1) binds the less-conserved consensus branchpoint sequence (CURAY) in human introns with a lower level of specificity and affinity. To determine which amino acids in BBP provide the additional specificity and affinity absent in SF1, a panel of chimeric SF1 proteins was tested in RNA binding assays with wild-type and mutant RNA substrates. This approach revealed that the QUA2 domain of BBP is responsible for the enhanced RNA binding affinity and specificity displayed by BBP compared with SF1. Within the QUA2 domain, a transposition of adjacent arginine and lysine residues is primarily responsible for the switch in RNA binding between BBP and SF1. Alignment of multiple branchpoint binding proteins and the related STAR/GSG proteins suggests that the identity of these two amino acids and the RNA target sequences of all of these proteins are correlated.