The two guard cells of a stoma are produced by a single symmetric division just before terminal differentiation. Recessive mutations in the FOUR LIPS (FLP) gene abnormally induce at least four guard cells in contact with one another. These pattern defects result from a persistence of precursor cell identity that leads to extra symmetric divisions at the end of the cell lineage. FLP is likely to be required for the correct timing of the transition from cell cycling to terminal differentiation. FLP encodes a two-repeat (R2R3) MYB protein whose expression accumulates just before the symmetric division. A paralogous gene, MYB88, overlaps with FLP function in generating normal stomatal patterning. Plants homozygous for mutations in both genes exhibit more severe defects than flp alone, and transformation of flp plants with a genomic MYB88 construct restores a wild-type phenotype. Both genes compose a distinct and relatively basal clade of atypical R2R3 MYB proteins that possess an unusual pattern of amino acid substitutions in their putative DNA binding domains. Our results suggest that two related transcription factors jointly restrict divisions late in the Arabidopsis thaliana stomatal cell lineage.

a b s t r a c tFor an enzyme functioning predominantly in a seemingly housekeeping role of 5 0 tRNA maturation, RNase P displays a remarkable diversity in subunit make-up across the three domains of life. Despite the protein complexity of this ribonucleoprotein enzyme increasing dramatically from bacteria to eukarya, the catalytic function rests with the RNA subunit during evolution. However, the recent demonstration of a protein-only human mitochondrial RNase P has added further intrigue to the compositional variability of this enzyme. In this review, we discuss some possible reasons underlying the structural diversity of the active sites, and use them as thematic bases for elaborating new directions to understand how functional variations might have contributed to the complex evolution of RNase P.

To the mounting evidence of nonribosomal functions for ribosomal proteins, we now add L7Ae as a subunit of archaeal RNase P, a ribonucleoprotein (RNP) that catalyzes 5′-maturation of precursor tRNAs (pre-tRNAs). We first demonstrate that L7Ae coelutes with partially purified Methanococcus maripaludis (Mma) RNase P activity. After establishing in vitro reconstitution of the single RNA with four previously known protein subunits (POP5, RPP21, RPP29, and RPP30), we show that addition of L7Ae to this RNase P complex increases the optimal reaction temperature and k cat ∕K m (by ∼360-fold) for pre-tRNA cleavage to those observed with partially purified native Mma RNase P. We identify in the Mma RNase P RNA a putative kink-turn (K-turn), the structural motif recognized by L7Ae. The large stimulatory effect of Mma L7Ae on RNase P activity decreases to ≤4% of wild type upon mutating either the conserved nucleotides in this K-turn or amino acids in L7Ae shown to be essential for K-turn binding. The critical, multifunctional role of archaeal L7Ae in RNPs acting in tRNA processing (RNase P), RNA modification (H/ACA, C/D snoRNPs), and translation (ribosomes), especially by employing the same RNA-recognition surface, suggests coevolution of various translation-related functions, presumably to facilitate their coordinate regulation.pre-tRNA processing | RPP38 | protein-aided RNA catalysis R Nase P is a Mg 2þ -dependent endoribonuclease that is primarily responsible for catalyzing the removal of the 5′ leaders of precursor-tRNAs (pre-tRNAs) (1-3). Except for some unique organellar variants, RNase P functions in all three domains of life as a ribonucleoprotein (RNP) (1, 2). Although catalysis rests with the essential RNase P RNA (RPR) in all three domains of life (4-6), the RNase P protein (RPP) cofactors play essential roles. In the simple one RPR-one RPP bacterial RNase P, the RPP aids RPR catalysis by enhancing cleavage efficiency and affinity for substrate and Mg 2þ (7-9). The bacterial RPP has not been found in any archaeal or eukaryal genome (10). Eukaryal (nuclear) RNase P, which comprises an RPR and 9 or 10 RPPs (11, 12), has not been reconstituted from recombinant subunits, thus thwarting efforts to uncover the individual functions of eukaryal RPPs. Archaeal RNase P, with an RPR and four RPPs (all homologous to eukaryal RPPs), has therefore been explored as an experimental surrogate for its so-far-intractable eukaryal cousin (13-16). Although native archaeal RNase P has not been characterized, Western analysis and immunoprecipitation validated these four RPPs (POP5, RPP21, RPP29, and RPP30) as being associated with partially purified Methanothermobacter thermautotrophicus (Mth) RNase P activity (14). Subsequent structural and biochemical reconstitution studies using recombinant subunits have proven the utility of archaeal RNase P as a model system to dissect the role of multiple protein cofactors in facilitating RNA catalysis (16).Besides POP5, RPP21, RPP29, and RPP30, weak homologies are evident in the archaeal genomes...

c Calcium carbonate increases growth, substrate utilization, and acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii NCIMB 8052. Toward an understanding of the basis for these pleiotropic effects, we profiled changes in the C. beijerinckii NCIMB 8052 proteome that occur in response to the addition of CaCO 3 . We observed increases in the levels of different heat shock proteins (GrpE and DnaK), sugar transporters, and proteins involved in DNA synthesis, repair, recombination, and replication. We also noted significant decreases in the levels of proteins involved in metabolism, nucleic acid stabilization, sporulation, oxidative and antibiotic stress responses, and signal transduction. We determined that CaCO 3 enhances ABE fermentation due to both its buffering effects and its ability to influence key cellular processes, such as sugar transport, butanol tolerance, and solventogenesis. Moreover, activity assays in vitro for select solventogenic enzymes revealed that part of the underpinning for the CaCO 3 -mediated increase in the level of ABE fermentation stems from the enhanced activity of these catalysts in the presence of Ca 2؉ . Collectively, these proteomic and biochemical studies provide new insights into the multifactorial basis for the stimulation of ABE fermentation and butanol tolerance in the presence of CaCO 3 . Growing concerns over increased emissions of greenhouse gases from the combustion of fossil fuels and the global energy crisis have recently spawned extensive research into renewable energy. As a result, there is a resurgent interest in butanol as an alternative fuel, due mainly to its higher energy content than ethanol and its compatibility with gasoline, with the latter trait making it more compatible with existing pipelines for distribution (1, 2). However, the cost of butanol production, which currently relies on petroleum feedstock, is not favorable compared to gasoline (3). Although acetone-butanol-ethanol (ABE) fermentation with solventogenic Clostridium species holds promise as a potentially cheaper means of butanol production, low yields and productivity due to butanol toxicity to the fermenting cells have hampered the commercialization of biobutanol (4, 5).To increase yield and productivity, fermentation broth additives such as acetate (6, 7) and calcium carbonate (3, 8, 9) have been successfully utilized. During ABE fermentation by solventogenic Clostridium species, CaCO 3 has been shown to stimulate sugar utilization, butanol production, and butanol tolerance (3,8,9). For example, during Clostridium acetobutylicum fermentation, the addition of 8 g/liter butanol (to mimic solvent intolerance) limited xylose utilization to 30 g/liter (from a starting concentration of 60 g/liter); however, upon the addition of CaCO 3 (10 g/liter), xylose utilization increased to 43 g/liter (8). Similarly, when ABE fermentation was conducted in an iron-deficient medium, which modifies carbon and electron flow to favor early butanol accumulation, the xylose utilization by C. acetobutyl...

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5′ leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533-1540. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5′ tRNA leaders. The "missing" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5′ pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5′ tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.catalytic RNA | gene finding | RNA processing R Nase P is best known for its role in removing the 5′ leaders of pre-tRNAs, an essential step in tRNA maturation. It also processes other RNAs in bacteria and eukaryotes, but these roles are less understood (1-3). RNase P typically functions as an RNA-protein complex, comprised of one conserved RNA and a varying number of protein subunits, depending on the domain of life: one in Bacteria, at least four in Archaea, and nine or more in the eukaryotic nucleus (4, 5). A precedent in which the RNA component is missing entirely is found in human and Arabidopsis organellar RNase P (6, 7), although a recent study suggests the possible coexistence of protein-only and RNA-protein-based RNase P complexes in human mitochondria (8).The inability to identify RNase P in some organisms has sown doubts about whether it is a universal feature of life. Studies of the hyperthermophilic bacterium Aquifex aeolicus showed that it exhibits RNase P-like trimming of tRNAs (9, 10), yet a gene for the expected protein component is absent and the RNA has remained elusive (11), prompting speculation that it may have developed a unique solution for pre-tRNA processing (9). Perhaps most surprisingly, Söll and coworkers (12) demonstrated that the archaeal symbiont Nanoarchaeum equ...