RNA regulators in bacteria are a heterogenous group of molecules that act by various mechanisms to modulate a wide range of physiological responses. One class comprises riboswitches, which are parts of the mRNAs they regulate. These leader sequences fold into structures amenable to conformational changes upon the binding of small molecules. Riboswitches thus sense and respond to the availability of various nutrients in the cell. Other small transcripts bind to proteins, among them global regulators, and antagonize their functions. The largest and most extensively studied set of small RNA regulators act through base pairing with RNAs, usually modulating the translation and stability of mRNAs. The majority of these small RNAs regulate responses to changes in environmental conditions. Finally, a recently discovered group of RNA regulators, known as the CRISPR RNAs, contain short regions of homology to bacteriophage and plasmid sequences. CRISPR RNAs interfere with bacteriophage infection and plasmid conjugation by targeting the homologous foreign DNA through an unknown mechanism. Here we discuss what is known about these RNA regulators, as well as the many intriguing questions that remain to be addressed.

SUMMARY DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol ζ, Pol κ, Pol η, and Pol ι.

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase (Rev3͞7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the Cterminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are Ϸ50-fold higher in G2 and throughout mitosis than during G1 and much of S phase. Differential survival of a rev1⌬ strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.cell cycle ͉ mutagenesis ͉ translesion synthesis ͉ DNA damage

Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action.

SUMMARY The highly-structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitch whose cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests the genes that are preceded by this motif, and encode a diverse set of poorly characterized membrane proteins, have roles in metal homeostasis.

Escherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.

The genes encoding Rev1 and DNA polymerase ζ (Rev3/Rev7) are together required for the vast majority of DNA damage-induced mutations in eukaryotes from yeast to humans. Here, we provide insight into the critical role that the Saccharomyces cerevisiae Rev1 C-terminus plays in the process of mutagenic DNA damage tolerance. The Rev1 C-terminus was previously thought to be poorly conserved and therefore not likely to be important for mediating protein-protein interactions. However, through comprehensive alignments of the Rev1 C-terminus, we have identified novel and hitherto unrecognized conserved motifs that we show play an essential role in REV1-dependent survival and mutagenesis in S. cerevisiae, likely in its post-replicative gap filling mode. We further show that the minimal C-terminal fragment of Rev1 containing these highly conserved motifs is sufficient to interact with Rev7.

Manganese is an essential trace nutrient for organisms, because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely-distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016 and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and ICP-MS to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negativelycharged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch, and found that the UPF0016 family of proteins has manganese export activity. We provide the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mnresponsive riboswitch can be used to identify new components of the manganese homeostasis machinery. INTRODUCTIONTransition metals are essential for life as they play important roles as enzyme cofactors and structural components of proteins and RNAs. Reflecting this, one third of the proteomes of organisms from bacteria to humans consist of metalloproteins (1,2). In bacteria, metal availability is intimately involved in pathogenesis. Bacteria unable to maintain proper metal homeostasis are less virulent, and mammalian hosts actively seek to withhold essential metals from invading bacteria (3,4). Yet in excess, metals are toxic to cells. This toxicity typically results from metaldependent oxidative damage (e.g., the Fenton reaction) and/or the displacement of cognate metals from their binding sites by the metal that is in excess (3,(5)(6)(7)(8). Thus, cells have a battery of metal importers, exporters, sequestration factors, and regulators to carefully control the intracellular level of each metal (1,2,9,10).