RNase R is a processive, 3 to 5 hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3 overhang is present. Using a variety of specifically designed substrates, we show here that a 3 overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5 overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3 to 5 polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3 termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA. The action of ribonucleases (RNases)2 is central to RNA metabolic processes such as the maturation of RNA precursors, the end-turnover of RNAs, and the degradation of RNAs that are either defective or are no longer required by the cell. Complete degradation of an RNA typically requires endoribonucleolytic cleavages followed by the action of a nonspecific 3Ј to 5Ј processive exoribonuclease (1). In Escherichia coli there are three such exoribonucleases: RNase II, RNase R, and polynucleotide phosphorylase (PNPase). RNase II and PNPase were originally thought to be responsible for mRNA decay (2). However, recent work has shown that mRNAs with extensive secondary structure, such as those containing repetitive extragenic palindromic sequences, are degraded by PNPase or by RNase R (3). Likewise, PNPase and/or RNase R is required for the degradation of rRNA (4) and tRNA (5), 3 both of which are highly structured molecules. These data suggest that PNPase and RNase R are the universal degraders of structured RNAs in vivo.Despite its role in the degradation of structured RNAs, purified PNPase is unable to digest through extensive secondary structure and stalls 6 -8 nucleotides (nt) before a stable RNA duplex (3). However, in vivo PNPase associates with an RNA helicase, RhlB, in the degradosome, which also contains the endoribonuclease, RNase E, and the glycolytic enzyme, enolase (6). A direct interaction with RhlB in the form of an ␣ 3 ␤ 2 complex has also been reported (7,8). It is likely to be the association with this helicase that allows PNPase to degrade through structure...

Control of cellular dimensions and cell symmetry are critical for development and differentiation. Here we provide evidence that the putative Rho-GAP Rga4p of Schizosaccharomyces pombe controls cellular dimensions. rga4 Delta cells are wider in diameter and shorter in length, whereas Rga4p overexpression leads to reduced diameter of the growing cell tip. Consistent with a negative role in cell growth control, Rga4p protein localizes to the cell sides in a "corset" pattern, and to the nongrowing cell tips. Additionally, rga4 Delta cells show an altered growth pattern similar to that observed in mutants of the formin homology protein For3p. Consistent with these observations, Rga4p is required for normal localization of For3p and for normal distribution of the actin cytoskeleton. We show that different domains of the Rga4p protein mediate diverse morphological functions. The C-terminal GAP domain mediates For3p localization to the cell tips and maintains cell diameter. Conversely, overexpression of the N-terminal LIM homology domain of Rga4p promotes actin cable formation in a For3p-dependent manner. Our studies indicate that Rga4p functionally interacts with For3p and has a novel function in the control of cell diameter and cell growth.

RNase II is a member of the widely distributed RNR family of exoribonucleases, which are highly processive 3'-->5' hydrolytic enzymes that play an important role in mRNA decay. Here, we report the crystal structure of E. coli RNase II, which reveals an architecture reminiscent of the RNA exosome. Three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single-stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single-stranded RNA.

RNase R and RNase II are the two representatives from the RNR family of processive, 3 to 5 exoribonucleases in Escherichia coli. Although RNase II is specific for single-stranded RNA, RNase R readily degrades through structured RNA. Furthermore, RNase R appears to be the only known 3 to 5 exoribonuclease that is able to degrade through double-stranded RNA without the aid of a helicase activity. Consequently, its functional domains and mechanism of action are of great interest. Using a series of truncated RNase R proteins we show that the cold-shock and S1 domains contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, whereas the S1 domain is most likely required to position substrates for efficient catalysis. Most importantly, the nuclease domain alone, devoid of the cold-shock and S1 domains, is sufficient for RNase R to bind and degrade structured RNAs. Moreover, this is a unique property of the nuclease domain of RNase R because this domain in RNase II stalls as it approaches a duplex. We also show that the nuclease domain of RNase R binds RNA more tightly than the nuclease domain of RNase II. This tighter binding may help to explain the difference in catalytic properties between RNase R and RNase II.Ribonucleases (RNases) play important roles in RNA metabolism. They are responsible for the maturation of stable RNA and the degradation of RNA molecules that are defective or no longer required by the cell. Both maturation and degradation are initiated by endoribonucleolytic cleavage(s) and completed by the action of exoribonucleases (1). In Escherichia coli, three, relatively nonspecific, 3Ј to 5Ј processive exoribonucleases are responsible for degradation of RNA: RNase II, RNase R, and polynucleotide phosphorylase (PNPase).3 RNase II and PNPase appear to be primarily responsible for mRNA decay (2), although their precise functions may differ (3). However, mRNAs containing extensive secondary structure, such as repetitive extragenic palindromic sequences, are degraded by PNPase (4, 5) or RNase R (5). Likewise, degradation of highly structured regions of rRNA (6) and tRNA (7), 4 is carried out by PNPase and/or RNase R. These findings suggest that PNPase and RNase R are the universal degraders of structured RNAs in vivo, leaving RNase II to act on relatively unstructured RNAs.Whether or not an RNase acts upon a particular RNA appears to depend upon the specificity of the RNase and the accessibility of the RNA to that RNase (1). Purified RNase R readily degrades both single-and double-stranded RNA molecules (5, 8), and it is the only known 3Ј to 5Ј exoribonuclease able to degrade through double-stranded RNA without the aid of helicase activity. To degrade RNA molecules containing double-stranded regions, RNase R requires a 3Ј single-stranded overhang at least 5 nucleotides long to serve as a binding site from which degradation can be initiated (5, 8, 9).5 How RNase R then proceeds through the RNA duplex is of great interest. An important step toward elucidating the ...

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.