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SreA is one of seven candidate S -adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes , a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenes may "dampen" the expression of genes for methionine import but likely does not turn them "OFF".

Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.

Protein production by ribosomes is fundamental to life, and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally modified, provides the platform for ribosome assembly. Thus, a complete understanding of ribosome assembly requires the determination of the RNA post-transcriptional modifications in all of the ribosome assembly intermediates and on each pathway. There are 26 RNA post-transcriptional modifications in 23S RNA of the mature Escherichia coli ( E. coli ) large ribosomal subunit. The levels of these modifications have been investigated extensively only for a small number of large subunit intermediates and under a limited number of cellular and environmental conditions. In this study, we determined the level of incorporations of 2-methyl adenosine, 3-methyl pseudouridine, 5-hydroxycytosine, and seven pseudouridines in an early-stage E. coli large-subunit assembly intermediate with a sedimentation coefficient of 27S. The 27S intermediate is one of three large subunit intermediates accumulated in E. coli cells lacking the DEAD-box RNA helicase DbpA and expressing the helicase inactive R331A DbpA construct. The majority of the investigated modifications are incorporated into the 27S large subunit intermediate to similar levels to those in the mature 50S large subunit, indicating that these early modifications or the enzymes that incorporate them play important roles in the initial events of large subunit ribosome assembly.

More than 55 distinct classes of riboswitches that respond to small metabolites or elemental ions have been experimentally validated to date. The ligands sensed by these riboswitches are biased in favor of fundamental compounds or ions that are likely to have been relevant to ancient forms of life, including those that might have populated the "RNA World", which is a proposed biochemical era that predates the evolutionary emergence of DNA and proteins. In the following text, I discuss the various types of ligands sensed by some of the most common riboswitches present in modern bacterial cells and consider implications for ancient biological processes centered on the proven capabilities of these RNA-based sensors. Although most major biochemical aspects of metabolism are represented by known riboswitch classes, there are striking sensory gaps in some key areas. These gaps could reveal weaknesses in the performance capabilities of RNA that might have hampered RNA World evolution, or these could highlight opportunities to discover additional riboswitch classes that sense essential metabolites.

An intriguing consequence of ongoing riboswitch discovery efforts is the occasional identification of metabolic or toxicity response pathways for unusual ligands. Recently, we reported the experimental validation of three distinct bacterial riboswitch classes that regulate gene expression in response to the selective binding of a guanidinium ion. These riboswitch classes, called guanidine-I, -II, and -III, regulate numerous genes whose protein products include previously misannotated guanidine exporters and enzymes that degrade guanidine via an initial carboxylation reaction. Guanidine is now recognized as the primal substrate of many multidrug efflux pumps that are important for bacterial resistance to certain antibiotics. Guanidine carboxylase enzymes had long been annotated as urea carboxylase enzymes but are now understood to participate in guanidine degradation. Herein, we report the existence of a fourth riboswitch class for this ligand, called guanidine-IV. Members of this class use a novel aptamer to selectively bind guanidine and use an unusual expression platform arrangement that is predicted to activate gene expression when ligand is present. The wide distribution of this abundant riboswitch class, coupled with the striking diversity of other guanidine-sensing RNAs, demonstrates that many bacterial species maintain sophisticated sensory and genetic mechanisms to avoid guanidine toxicity. This finding further highlights the mystery regarding the natural source of this nitrogen-rich chemical moiety.

RNA thermosensors (RNATs), found in the 5' untranslated region (UTR) of some bacterial messenger RNAs (mRNAs), control the translation of the downstream gene in a temperature-dependent manner. In Listeria monocytogenes , the expression of a key transcription factor, PrfA, is mediated by an RNAT in its 5' UTR. PrfA functions as a master regulator of virulence in L. monocytogenes , controlling the expression of many virulence factors. The temperature-regulated expression of PrfA by its RNAT element serves as a signal of successful host invasion for the bacteria. Structurally, the prfA RNAT bears little resemblance to known families of RNATs, and prior studies demonstrated that the prfA RNAT is highly responsive over a narrow temperature range. Herein, we have undertaken a comprehensive mutational and thermodynamic analysis to ascertain the molecular determinants of temperature sensitivity. We provide evidence to support the idea that the prfA RNAT unfolding is different from that of cssA, a well-characterized RNAT, suggesting that these RNATs function via distinct mechanisms. Our data show that the unfolding of the prfA RNAT occurs in two distinct events and that the internal loops play an important role in mediating the cooperativity of RNAT unfolding. We further demonstrated that regions distal to the ribosome binding site (RBS) not only contribute to RNAT structural stability but also impact translation of the downstream message. Our collective results provide insight connecting the thermal stability of the prfA RNAT structure, unfolding energetics, and translational control.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λP R promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λP R . Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d. Subtype 2a RNAs are riboswitches that sense the bacterial alarmone ppGpp and typically regulate amino acid biosynthesis genes. Subtype 2b riboswitches sense the purine biosynthetic intermediate PRPP and frequently partner with guanine riboswitches to regulate purine biosynthesis genes. In this study, we examined ykkC subtype 2c RNAs, which are found upstream of genes encoding hydrolase enzymes that cleave the phosphoanhydride linkages of nucleotide substrates. Subtype 2c representatives mostly recognize adenosine and cytidine 5'-diphosphate molecules in either their ribose or deoxyribose forms (ADP, dADP, CDP, and dCDP). Other nucleotide-containing compounds, especially nucleoside 5'-triphosphates, are strongly rejected by some members of this putative riboswitch class. High ligand concentrations in vivo are predicted to turn on expression of hydrolase enzymes, which presumably function to balance cellular nucleotide pools. These results further showcase the striking functional diversity derived from the structural scaffold shared among all ykkC motif RNAs, which has been adapted to sense at least five different types of natural ligands. Moreover, riboswitches for nucleoside diphosphates provide additional examples of the numerous partnerships observed between natural RNA aptamers and nucleotide-derived ligands, including metabolites, coenzymes, and signaling molecules.

RNA is a key player in the cellular central dogma, including RNA transcription and protein synthesis. However, it is unknown whether RNA can directly interfere with DNA synthesis. Recently, we have found in vitro that while binding to DNA polymerase nonspecifically, RNA can transform DNA polymerase to display a moonlighting activity, dNTP phosphatase, in turn interfering with DNA synthesis. This phosphatase activity removes the γ-phosphate from dNTPs (generating dNDPs) and subsequently removes the β-phosphate from the formed dNDPs (generating dNMPs), confirmed by the noncleavable α,β-CH 2 -dGTP and β,γ-CH 2 -dGTP analogues. We also found that dGTP is the best substrate for the phosphatase, and the dNTP phosphatase activity is sensitive to the reaction medium. In addition, we have revealed that RNA can tune the activity of closely related proteins and give rise to new catalytic functions with subtle differences. Moreover, we have demonstrated in vitro that at the lower dNTP level, this phosphatase can directly inhibit DNA synthesis by dNTP depletion, though the phosphatase activity is 690-fold slower than the polymerase activity. Our observation in vitro suggests a plausible strategy for RNA to directly interfere with DNA polymerase and DNA synthesis in vivo.

A complete understanding of the determinants that restrict d-amino acid incorporation by the ribosome, which is of interest to both basic biologists and the protein engineering community, remains elusive. Previously, we demonstrated that d-amino acids are successfully incorporated into the C-terminus of the nascent polypeptide chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA (peptidyl-d-aa-tRNA) donor substrate, however, partition into subpopulations that either undergo translation arrest through inactivation of the ribosomal peptidyl-transferase center (PTC) or remain translationally competent. The proportion of each subpopulation is determined by the identity of the d-amino acid side chain. Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA) acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery demonstrates that it is the pairing of the peptidyl-d-aa-tRNA donor and the aa-tRNA acceptor that determines the activity of the PTC. Moreover, we provide evidence that both the amino acid and tRNA components of the aa-tRNA acceptor contribute synergistically to the extent of arrest. The results of this work deepen our understanding of the mechanism of d-amino acid-mediated translation arrest and how cells avoid this precarious obstacle, reveal similarities to other translation arrest mechanisms involving the PTC, and provide a new route for improving the yields of engineered proteins containing d-amino acids.

The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron-sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human l-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of ∼60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm 5 s 2 U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.

In Gram-positive bacteria, the tRNA-dependent T-box riboswitch system regulates expression of amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. Binding of uncharged tRNA "closes" the switch, allowing transcription read-through. Structural studies of the 100-nucleotide stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180-nucleotide riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA or the mechanism of coupling of the intermolecular binding domains crucial to T-box function. Here we utilize solution structural and biophysical methods to characterize the interplay of the different riboswitch-tRNA contact points using Bacillus subtilis and Oceanobacillus iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier-anticodon and antiterminator-acceptor stem pairing. The elbow:platform stacking interaction observed in studies of the T-box stem I domain is released after pairing between the acceptor stem and the bulge in the antiterminator helix. The results are consistent with the model of T-box riboswitch:tRNA function in which tRNA is captured by stem I of the nascent mRNA followed by stabilization of the antiterminator helix and the paused transcription complex.

Recently, it was determined that representatives of the riboswitch candidates called ykkC and ykkC-III directly bind free guanidine. Guanidine-binding ykkC motif RNAs, now renamed guanidine-I riboswitches, were demonstrated to commonly regulate the expression of genes encoding guanidine carboxylases, as well as others encoding guanidine efflux proteins such as EmrE and SugE. Likewise, genes encoding similar efflux proteins are associated with ykkC-III motif RNAs, which have now been renamed guanidine-III riboswitches. Prior to the validation of guanidine as the ligand for these newly established riboswitch classes, another RNA motif was discovered by comparative genomic analysis and termed mini-ykkC because of its small size and gene associations similar to those of the original ykkC motif. It was hypothesized that these distinct RNA structures might respond to the same ligand. However, the small size and repetitive nature of mini-ykkC RNAs suggested that it might respond to ligand via the action of a protein factor. Herein, we demonstrate that, despite its extremely simple architecture, mini-ykkC motif RNAs constitute a distinct class of guanidine-sensing RNAs, called guanidine-II riboswitches. Surprisingly, each of the two stem-loop structures that comprise the mini-ykkC motif appears to directly bind free guanidine in a cooperative manner. These findings reveal that bacteria make extensive use of diverse guanidine-responsive riboswitches to overcome the toxic effects of this compound.

Recently, it was determined that representatives of the riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II riboswitch RNAs, respectively. Moreover, we had previously noted that a third candidate riboswitch class, called ykkC-III, was associated with a distribution of genes similar to those of the other two motifs. Therefore, it was predicted that ykkC-III motif RNAs would sense and respond to the same ligand. In this report, we present biochemical data supporting the hypothesis that ykkC-III RNAs represent a third class of guanidine-sensing RNAs called guanidine-III riboswitches. Members of the guanidine-III riboswitch class bind their ligand with an affinity similar to that observed for members of the other two classes. Notably, there are some sequence similarities between guanidine-II and guanidine-III riboswitches. However, the characteristics of ligand discrimination by guanidine-III RNAs are different from those of the other guanidine-binding motifs, suggesting that the binding pockets have distinct features among the three riboswitch classes.

The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3'-5'-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways.

Transcription elongation requires the continuous incorporation of ribonucleotide triphosphates into a growing transcript. RNA polymerases (RNAPs) are able to processively synthesize a growing RNA chain via translocation of the RNAP enzyme along its nucleic acid template strand after each nucleotide addition cycle. In this work, a time-resolved Raman spectroscopic analysis of nucleotide addition in single crystals of the Thermus thermophilus elongation complex (TthEC) is reported. When [(13)C,(15)N]GTP (*GTP) is soaked into crystals of the TthEC, large reversible changes in the Raman spectrum that are assigned to protein and nucleic acid conformational events during a single-nucleotide incorporation are observed. The *GTP population in the TthEC crystal reaches a stable population at 37 min, while substantial and reversible protein conformational changes (mainly ascribed to changes in α-helical Raman features) maximize at approximately 50 min. At the same time, changes in nucleic acid bases and phosphodiester backbone Raman marker bands occur. Catalysis begins at approximately 65-70 min, soon after the maximal protein and DNA changes, and is monitored via the decline in a triphosphate vibrational Raman mode from *GTP. The Raman data indicate that approximately 40% of the total triphosphate population, present as *GTP, reacts in the crystal. This may suggest that a second population of noncovalently bound *GTP resides in a site distinct from the catalytic site. The data reported here are an extension of our recent work on the elongation complex (EC) of a bacterial RNAP, Thermus thermophilus (Tth), where Raman spectroscopy and polyacrylamide gel electrophoresis were employed to monitor incorporation and misincorporation in single TthEC crystals [Antonopoulos, I. H., et al. (2015) Biochemistry 54, 652-665]. Therefore, the initial study establishes the groundwork for this study. In contrast to our previous study, in which incorporation takes place very rapidly inside the crystals, the data on this single crystal exhibit a slower time regime, which allows the dissection of the structural dynamics associated with GMP incorporation within the TthEC crystal.

In an earlier study, β³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA(CUA) activated with each of four methyl-β-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated β-tyrosine moiety of β³-puromycin. Also conducted were a selection of clones that are responsive to β²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.

Bacterial regulatory RNAs require the chaperone protein Hfq to enable their pairing to mRNAs. Recent data showed that there is a hierarchy among sRNAs in the competition for access to Hfq, which could be important for the tuning of sRNA-dependent translation regulation. Here, seven structurally different sRNAs were compared using filter-based competition assays. Moreover, chimeric sRNA constructs were designed to identify structure elements important for competition performance. The data showed that besides the 3'-terminal oligouridine sequences also the 5'-terminal structure elements of sRNAs were essential for their competition performance. When the binding of sRNAs to Hfq mutants was compared, the data showed the important role of the proximal and rim sites of Hfq for the binding of six out of seven sRNAs. However, ChiX sRNA, which was the most efficient competitor, bound Hfq in a unique way using the opposite-distal and proximal-faces of this ring-shaped protein. The data indicated that the simultaneous binding to the opposite faces of Hfq was enabled by separate adenosine-rich and uridine-rich sequences in the long, single-stranded region of ChiX. Overall, the results suggest that the individual structural composition of sRNAs serves to tune their performance to different levels resulting in a hierarchy of sRNAs in the competition for access to the Hfq protein.

The bacterial RNA polymerase (RNAP) elongation complex (EC) is highly stable and is able to extend an RNA chain for thousands of nucleotides. Understanding the processive mechanism of nucleotide addition requires detailed structural and temporal data for the EC reaction. Here, a time-resolved Raman spectroscopic analysis is combined with polyacrylamide gel electrophoresis (PAGE) to monitor nucleotide addition in single crystals of the Thermus thermophilus EC (TthEC) RNAP. When the cognate base GTP, labeled with (13)C and (15)N (*GTP), is soaked into crystals of the TthEC, changes in the Raman spectra show evidence of nucleotide incorporation and product formation. The major change is the reduction of *GTP's triphosphate intensity. Nucleotide incorporation is confirmed by PAGE assays. Both Raman and PAGE methods have a time resolution of minutes. There is also Raman spectroscopic evidence of a second population of *GTP in the crystal that does not become covalently linked to the nascent RNA chain. When this population is removed by "soaking out" (placing the crystal in a solution that contains no NTP), there are no perturbations to the Raman difference spectra, indicating that conformational changes are not detected in the EC. In contrast, the misincorporation of the noncognate base, (13)C- and (15)N-labeled UTP (*UTP), gives rise to large spectroscopic changes. As in the GTP experiment, reduction of the triphosphate relative intensity in the Raman soak-in data shows that the incorporation reaction occurs during the first few minutes of our instrumental dead time. This is also confirmed by PAGE analysis. Whereas PAGE data show *GTP converts 100% of the nascent RNA 14mer to 15mer, the noncognate *UTP converts only ∼50%. During *UTP soak-in, there is a slow, reversible formation of an α-helical amide I band in the Raman difference spectra peaking at 40 min. Similar to *GTP soak-in, *UTP soak-in shows Raman spectoscopic evidence of a second noncovalently bound *UTP population in the crystal. Moreover, the second population has a marked effect on the complex's conformational states because removing it by "soaking-out" unreacted *UTP causes large changes in protein and nucleic acid Raman marker bands in the time range of 10-100 min. The conformational changes observed for noncognate *UTP may indicate that the enzyme is preparing for proofreading to excise the misincorporated base. This idea is supported by the PAGE results for *UTP soak-out that show endonuclease activity is occurring.

In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA-protein complexes. We used single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of Escherichia coli 16S rRNA in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base-paired structure different from that expected on the basis of comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse-chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to preorganize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes.

HDV-like self-cleaving ribozymes have been found in a wide variety of organisms, implicated in diverse biological processes, and their activity typically shows a strong divalent metal dependence, but little metal specificity. Recent studies suggested that very short variants of these ribozymes exist in nature, but their distribution and biochemical properties have not been established. To map out the distribution of small HDV-like ribozymes, the drz-Spur-3 sequence was minimized to yield a core construct for structure-based bioinformatic searches. These searches revealed several microbial ribozymes, particularly in the human microbiome. Kinetic profile of the smallest ribozyme revealed two distinct metal binding sites, only one of which promotes fast catalysis. Furthermore, this ribozyme showed markedly reduced activity in Ca(2+), even in the presence of physiological Mg(2+) concentrations. Our study substantially expands the number of microbial HDV-like ribozymes and provides an example of cleavage regulation by divalent metals.

The dynamic behavior of the rRNA A-site plays an important functional role. We have employed femtosecond time-resolved spectroscopy to investigate the nature of the conformational dynamics. In the drug-free state, the A-site samples multiple distinct conformations. Drug binding shifts the population distribution in a drug-specific manner. Motions of bases on nanosecond and picosecond time scales are differentially affected by the drug binding. Our results underscore the importance of understanding the detailed dynamic picture of molecular recognition by resolving dynamics in the distinct picosecond time regime and facilitate development of antimicrobial drugs targeting dynamic RNAs.

Shadoo (Sho) is a central nervous system glycoprotein with characteristics similar to those of the cellular prion protein PrP(C), each containing a highly conserved hydrophobic domain (HD) and an N-terminal repeat region. Whereas PrP(C) includes histidine-containing octarepeats, the Sho region N-terminal to the HD includes tandem positively charged "RGG boxes", predicted to bind RNA. Here, we demonstrate that Sho binds DNA and RNA in vitro via this arginine-rich region.

The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (K(D)) for lysine binding of 1.7(5) mM and opening/closing rate constants of 1.4(3) s(-1) and 0.203(7) s(-1), respectively. These rates predict an apparent dissociation constant for lysine binding (K(D,apparent)) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports.

In an RNA transcript, the 2'-OH group at the 3'-terminal nucleotide is unique as it is the only 2'-OH group that is adjacent to a 3'-OH group instead of a phosphate backbone. The 2'-OH group at the 3'-terminal nucleotide of certain RNAs is methylated in vivo, which is acheived by a methyltransferase named Hen1 that is mechanistically distinct from other known RNA 2'-O-methyltransferases. In eukaryotic organisms, 3'-terminal 2'-O-methylation of small RNAs stabilizes these small RNAs for RNA interference (RNAi). In bacteria, the same methylation during RNA repair results in repaired RNA resisting future damage at the site of repair. Although the chemistry performed by the eukaryotic and bacterial Hen1 is the same, the mechanisms of how RNA is stabilized as a result of the 3'-terminal 2'-O-methylation are different between the eukaryotic RNAi and the bacterial RNA repair. In this review, I will discuss the distribution of Hen1 in living organisms, the classification of Hen1 into four subfamilies, the structure and mechanism of Hen1 that allows it to conduct RNA 3'-terminal 2'-O-methylation, and the possible evolutionary origin of Hen1 present in bacterial and eukaryotic organisms.

Specific (13)C labeling of Thr methyl groups has been accomplished via the growth of a standard laboratory strain of Escherichia coli on [2-(13)C]glycerol in the presence of deuterated isoketovalerate, Ile, and Ala. Diversion of the label from the Thr biosynthetic pathway is suppressed by including Lys, Met, and Ile in the growth medium. This method complements the repertoire of methyl labeling schemes for NMR structural and dynamic studies of proteins and is particularly useful for the study of nucleic acid binding proteins because of the high propensity of Thr residues at protein-DNA and -RNA interfaces.

Information on the secondary structure and interactions of RNA is important to understand the biological function of RNA as well as in applying RNA as a tool for therapeutic purposes. Recently, the isoenergetic microarray mapping method was developed to improve the prediction of RNA secondary structure. Herein, for the first time, isoenergetic microarrays were used to study the binding of RNA to protein or other RNAs as well as the interactions of two different RNAs and protein in a three-component complex. The RNAs used as models were the regulatory DsrA and OxyS RNAs from Escherichia coli, the fragments of their target mRNAs (fhlA and rpoS), and their complexes with Hfq protein. The collected results showed the advantages and some limitations of microarray mapping.

The binding of nine noncoding regulatory RNAs (sRNAs) to the E. coli Hfq protein was compared using a high-throughput double filter retention assay. Despite the fact that these sRNAs have different lengths, sequences and secondary structures their Hfq binding affinities were surprisingly uniform. The analysis of sRNAs binding to Hfq mutants showed that the proximal face of Hfq, known as the binding site for DsrA RNA, is a universal sRNA binding site. Moreover, all sRNAs bound Hfq with similar association rates limited only by the rate of diffusion, while the rates of dissociation, measured in the dilution experiments, were uniformly slow. Despite that, the data showed that there was a hierarchy of sRNAs in regard to their performance in competition for access to Hfq and in their ability to facilitate the dissociation of other sRNAs from Hfq. The sRNAs also differed in their salt dependence of binding to this protein. Overall, the results suggest that despite the uniform binding of different sRNAs to the same site on Hfq their exchange on this protein is dependent on the identities of the competing sRNAs.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5' UTR of the glmS mRNA, resulting in downregulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg(2+)-containing buffers effectively isolates the rapid ligand binding and catalytic events (k(obs) > 60 min(-1)) from rate-limiting folding (k(obs) < 4 min(-1)). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure but are unable to promote ligand binding and catalysis rates of >4 min(-1). Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand-binding pocket. In further support of this hypothesis, our data show that a nonphosphorylated analogue of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity, but the rapid initiation of catalysis upon the addition of ligand is stricter.

The selection of aminoacyl-tRNAs by the ribosome is a fundamental step in the elongation cycle of protein synthesis. tRNA selection is a multistep process that ensures only correct aminoacyl-tRNAs are accepted while incorrect aminoacyl-tRNAs are rejected. A key step in tRNA selection is the formation of base pairs between the anticodon of the aminoacyl-tRNA and the mRNA codon in the A site, called "codon recognition". Here, we report the development of a new, fluorescence-based, kinetic assay for monitoring codon recognition by the ribosome. Using this assay, we show that codon recognition is a second-order binding step under optimal conditions. Additionally, we show that at low Mg(2+) concentrations, the polyamines spermine and spermidine stimulate codon recognition by the ribosome without a loss of fidelity. Polyamines may accelerate codon recognition by altering the structure and dynamics of the anticodon arm of the aminoacyl-tRNA.

The cleavage of double-stranded (ds) RNA by ribonuclease III is a conserved early step in bacterial rRNA maturation. Studies on the mechanism of dsRNA cleavage by RNase III have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, neither the catalytic properties of extremophile RNases III nor the structures and reactivities of their cognate substrates have been described. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima was analyzed using purified recombinant enzyme. T. maritima (Tm) RNase III catalytic activity exhibits a broad optimal temperature range of approximately 40-70 degrees C, with significant activity at 95 degrees C. Tm-RNase III cleavage of substrate is optimally supported by Mg(2+) at >or=1 mM concentrations. Mn(2+), Co(2+), and Ni(2+) also support activity but with reduced efficiencies. The enzyme functions optimally at pH 8 and approximately 50-80 mM salt concentrations. Small RNA hairpins that incorporate the 16S and 23S pre-rRNA stem sequences are efficiently cleaved by Tm-RNase III at sites that are consistent with production in vivo of the immediate precursors to the mature rRNAs. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive recognition determinant for Escherichia coli (Ec) RNase III substrates. The dependence of reactivity on the pb sequence is similar to that observed with Ec-RNase III substrates. In fact, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity and is inhibited by antideterminant bp that also inhibit Ec-RNase III. These results indicate the conservation, across a broad phylogenetic distance, of positive and negative determinants of reactivity of bacterial RNase III substrates.

RNAs have diverse structures that are important for biological function. These structures include bulges and internal loops that can form tertiary contacts or serve as ligand binding sites. The most commonly exploited RNA drug target for small molecule intervention is the bacterial ribosome, more specifically the rRNA aminoacyl-tRNA site (rRNA A-site) which is a major target for the aminoglycoside class of antibiotics. The bacterial A-site is composed of a 1 x 1 nucleotide all-U internal loop and a 2 x 1 nucleotide all-A internal loop separated by a single GC base pair. Therefore, we probed the molecular recognition of a small library of four aminoglycosides for binding a 16384-member bacterial rRNA A-site-like internal loop library using two-dimensional combinatorial screening (2DCS). 2DCS is a microarray-based method that probes RNA and chemical spaces simultaneously. These studies sought to determine if aminoglycosides select their therapeutic target if given a choice of binding all possible internal loops derived from an A-site-like library. Results show that the bacterial rRNA A-site was not selected by any aminoglycoside. Analyses of selected sequences using the RNA Privileged Space Predictor (RNA-PSP) program show that each aminoglycoside preferentially binds different types of internal loops. For three of the aminoglycosides, 6''-azido-kanamycin A, 5-O-(2-azidoethyl)-neamine, and 6''-azido-tobramycin, the selected internal loops bind with approximately 10-fold higher affinity than the bacterial rRNA A-site. The internal loops selected to bind 5''-azido-neomycin B bind with an affinity similar to that of the therapeutic target. Selected internal loops that are unique for each aminoglycoside have dissociation constants ranging from 25 to 270 nM and are specific for the aminoglycoside they was selected to bind compared to the other arrayed aminoglycosides. These studies further establish a database of RNA motifs that are recognized by small molecules that could be used to enable the rational and modular design of small molecules targeting RNA.

RapA, a prokaryotic member of the SWI/SNF protein superfamily, is an integral part of the RNA polymerase transcription complex. RapA's function and catalytic mechanism have been linked to nucleic acid remodeling. In this work, we show that mutations in the interface between RapA's SWI/SNF and double-stranded nucleic acid-binding domains significantly alter ATP hydrolysis in purified RapA. The effects of individual mutations on ATP hydrolysis loosely correlated with RapA's nucleic acid remodeling activity, indicating that the interaction between these domains may be important for the RapA-mediated remodeling of nonproductive transcription complexes. In this study, we introduced a model system for in vitro transcription of a full-length Escherichia coli gene (slyD). To study the function of RapA, we fractionated and identified in vitro transcription reaction intermediates in the presence or absence of RapA. These experiments demonstrated that RapA contributes to the formation of free RNA species during in vitro transcription. This work further refines our models for RapA function in vivo and establishes a new role in RNA management for a representative of the SWI/SNF protein superfamily.

Escherichia coli polynucleotide phosphorylase (PNPase) primarily functions in RNA degradation. It is an exoribonuclease and integral component of the multienzyme RNA degradosome complex [Carpousis et al. (1994) Cell 76, 889]. PNPase was previously shown to specifically bind a synthetic RNA containing the oxidative lesion 8-hydroxyguanine (8-oxoG) [Hayakawa et al. (2001) Biochemistry 40, 9977], suggesting a possible role in removing oxidatively damaged RNA. Here we show that PNPase binds to RNA molecules of natural sequence that were oxidatively damaged by treatment with hydrogen peroxide (H(2)O(2)) postsynthetically. PNPase bound oxidized RNA with higher affinity than untreated RNA of the same sequence, raising the possibility that it may act against a wide variety of lesions. The importance of such a protective role is illustrated by the observation that, under conditions known to cause oxidative damage to cytoplasmic components, PNPase-deficient cells are less viable than wild-type cells. Further, when challenged with H(2)O(2), PNPase-deficient cells accumulate 8-oxoG in cellular RNA to a greater extent than wild-type cells, suggesting that this RNase functions in minimizing oxidized RNA in vivo. Introducing the pnp gene encoding PNPase rescues defects in growth and RNA quality of the pnp mutant cells. Our results also suggest that protection against oxidative stress is an intrinsic function of PNPase because association with the RNA degradosome or with RNA helicase B (RhlB) is not required.

RTN1-C protein is a membrane protein localized in the ER and expressed in the nervous system. Its biological role is still unclear, although interactions of the N-terminal region of RTN1-C with proteins involved in vesicle trafficking have been observed, but the role of the C-terminal region of this family protein remains to be investigated. By a homology analysis of the amino acid sequence, we identified in the C-terminal region of RTN1-C a unique consensus sequence characteristic of H4 histone protein. Thus, a 23-mer peptide (RTN1-C(CT)) corresponding to residues 186-208 of RTN1-C was synthesized, and its conformation and its interaction with nucleic acids were investigated. Here we demonstrate the strong ability of RTN1-C(CT) peptide to bind and condense the nucleic acids using electrophoretic and spectroscopic techniques. To determine if the binding of RTN1-C to nucleic acids could be regulated in vivo by an acetylation-deacetylation mechanism, as for the histone proteins, we studied the interaction of RTN1-C with one zinc-dependent histone deacetylase (HDAC) enzyme, HDAC8, with fluorescence and kinetic techniques using an acetylated form of RTN1-C(CT). The results reported here allow us to propose that the nucleic acid binding property of RTN1-C may have an important role in the biological function of this protein, the function of which could be regulated by an acetylation-deacetylation mechanism.

The rpoS mRNA encodes a stress response transcription factor in Escherichia coli. It is one of a growing number of mRNAs found to be regulated by small RNAs (sRNA). Translation initiation of rpoS mRNA is enhanced by two sRNAs, DsrA and RprA, that pair to the same site near the rpoS start codon in the presence of the Hfq protein. In this work, we examine the interaction of E. coli Hfq with RprA and two portions of the rpoS mRNA leader region. One rpoS RNA, rpoS-L, contained the entire 565-nucleotide leader region, while the other, rpoS-S, contained the 199-nucleotide sequence surrounding the start codon. An RNase H assay indicated both rpoS RNAs have similar secondary structures in the translation initiation region. Hfq formed two complexes with RprA in a gel mobility assay with binding parameters similar to values previously determined for DsrA. Unlike DsrA, Hfq binding to RprA was inhibited by poly(A) and influenced by Hfq mutations on both the distal and proximal surfaces. Hfq increased the level of RprA binding to both rpoS RNAs but showed a much larger enhancement when rpoS-L, the entire leader region, was examined. The lower affinity of RprA for rpoS-L versus rpoS-S in the absence of Hfq suggests that Hfq overcomes an inhibitory structure within rpoS-L in stimulating RprA binding. Similar results were obtained with DsrA. The results indicate that the full upstream leader sequence of rpoS mRNA influences Hfq-facilitated annealing of RprA and DsrA and is likely to be involved in its regulation.

The interaction of tRNA and its carrier, exportin-t, was studied by photoaffinity cross-linking of randomly s (4)U-substituted tRNA (Phe) T.th in vitro transcript that was able to form complex with exportin-t and ran.GTP. We found that in the ternary complex, tRNA (Phe) T.th was cross-linked only to exportin-t, not to ran.GppNHp (an analogue of ran.GTP), suggesting that tRNA had much stronger contact with exportin-t than with ran.GTP. The contact sites between tRNA (Phe) T.th and exportin-t were supposed to be the cross-linking sites in this study and further determined by primer extension analysis. The major cross-linking sites were U55, U47, U20, and U33. Apart from the footprinting data on the contact sites between exportin-t and tRNA, which are mainly on the acceptor arm and TPsiC region, our results showed that exportin-t bound tRNA in a very extensive way, and the anticodon loop and D loop might also contact, though weakly, the tRNA carrier. Using the site-directed mutagenesis method, we also found that while position 47 in tRNA (Phe) T.th is important for the binding interaction, the identity of the nucleotide at this position is not.

Histone-like proteins (such as HU, H-NS, and Fis) participate in nucleoid organization and in DNA replication, recombination, and transcription. Cold shock and anoxia upregulates a homologue of HU (Hlp) in Mycobacterium smegmatis, the nonpathogenic model of Mycobacterium tuberculosis. We show using electrophoretic mobility shift assays that Hlp, which in addition to the HU fold has a basic C-terminal tail containing multiple PAKK and PAAK repeats, has very high affinity for DNA. The affinity of Hlp for 76 bp linear DNA is higher, K d = 0.037 +/- 0.001 nM, compared to an Hlp variant without the C-terminal repeats, K d = 2.5 +/- 0.1 nM and the isolated C-terminal repeat domain, K d = 0.8 +/- 0.2 nM, where K d in all cases reflects an aggregate affinity for the DNA probes, not the affinity for binding to a single site. Hlp lacking the entire C-terminal domain binds DNA only poorly. These data indicate that both Hlp domains contribute to high-affinity DNA binding. Hlp promotes DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At <100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when present together. Notably, while DNA end-joining can be achieved by the isolated C-terminal domain, transcriptional repression requires for both domains to be present on a single polypeptide. Given the low cellular concentration of Hlp, our data suggest that its primary functional role may be in DNA-dependent responses to environmental stress rather than in nucleoid organization.

Lacticin 481 is a lanthionine-containing bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. The final steps of lacticin 481 biosynthesis are proteolytic removal of an N-terminal leader sequence from the prepeptide LctA and export of the mature lantibiotic. Both proteolysis and secretion are performed by the dedicated ATP-binding cassette (ABC) transporter LctT. LctT belongs to the family of AMS (ABC transporter maturation and secretion) proteins whose prepeptide substrates share a conserved double-glycine type cleavage site. The in vitro activity of a lantibiotic protease has not yet been characterized. This study reports the purification and in vitro activity of the N-terminal protease domain of LctT (LctT150), and its use for the in vitro production of lacticin 481. The G(-2)A(-1) cleavage site and several other conserved amino acid residues in the leader peptide were targeted by site-directed mutagenesis to probe the substrate specificity of LctT as well as shed light upon the role of these conserved residues in lantibiotic biosynthesis. His 10-LctT150 did not process most variants of the double glycine motif and processed mutants of Glu-8 only very slowly. Furthermore, incorporation of helix-breaking residues in the leader peptide resulted in greatly decreased proteolytic activity by His 10-LctT150. On the other hand, His 10-LctT150 accepted all peptides containing mutations in the propeptide or at nonconserved positions of LctA. In addition, the protease domain of LctT was investigated by site-directed mutagenesis of the conserved residues Cys12, His90, and Asp106. The proteolytic activities of the resulting mutant proteins are consistent with a cysteine protease.

M1 RNA, the gene product of rnpB, is the catalytic subunit of RNase P in Escherichia coli. M1 RNA is transcribed from a proximal promoter as pM1 RNA, a precursor M1 RNA, and then is processed at its 3' end by RNase E. In addition to pM1 RNA, large rnpB-containing transcripts are produced from unknown upstream promoters. However, it is not known yet how these large transcripts contribute to M1 RNA biosynthesis. To examine their biological relevance to M1 RNA biosynthesis, we constructed a model upstream transcript, upRNA, and analyzed its cellular metabolism. We found that upRNA was primarily degraded rather than processed to M1 RNA in the cell and that this degradation occurred in RNase E-dependent manner. The in vitro cleavage assay with the N-terminal catalytic fraction of RNase E showed that the M1 RNA structural sequence in upRNA was much more vulnerable to the enzyme than the sequence in pM1 RNA. Considering that RNase E is a processing enzyme involved in 3' end formation of M1 RNA, our results imply that this enzyme plays a dual role in processing and degradation to achieve tight control of M1 RNA biosynthesis.

The purine riboswitch is one of a number of mRNA elements commonly found in the 5'-untranslated region capable of controlling expression in a cis-fashion via its ability to directly bind small-molecule metabolites. Extensive biochemical and structural analysis of the nucleobase-binding domain of the riboswitch, referred to as the aptamer domain, has revealed that the mRNA recognizes its cognate ligand using an intricately folded three-way junction motif that completely encapsulates the ligand. High-affinity binding of the purine nucleobase is facilitated by a distal loop-loop interaction that is conserved between both the adenine and guanine riboswitches. To understand the contribution of conserved nucleotides in both the three-way junction and the loop-loop interaction of this RNA, we performed a detailed mutagenic survey of these elements in the context of an adenine-responsive variant of the xpt-pbuX guanine riboswitch from Bacillus subtilis. The varying ability of these mutants to bind ligand as measured by isothermal titration calorimetry uncovered the conserved nucleotides whose identity is required for purine binding. Crystallographic analysis of the bound form of five mutants and chemical probing of their free state demonstrate that the identity of several universally conserved nucleotides is not essential for formation of the RNA-ligand complex but rather for maintaining a binding-competent form of the free RNA. These data show that conservation patterns in riboswitches arise from a combination of formation of the ligand-bound complex, promoting an open form of the free RNA, and participating in the secondary structural switch with the expression platform.

EF-G catalyzes translocation of mRNA and tRNAs within the ribosome during protein synthesis. Detection of structural states in the reaction sequence that are not highly populated can be facilitated by studying the process one molecule at a time. Here we present single-molecule studies of translocation showing that, for ribosomes engaged in poly(Phe) synthesis, fluorescence resonance energy transfer (FRET) between the G' domain of EF-G and the N-terminal domain of ribosomal protein L11 occurs within two rapidly interconverting states, having FRET efficiencies of 0.3 and 0.6. The antibiotic fusidic acid increases the population of the 0.6 state, indicating that it traps the ribosome.EF-G complex in a preexisting conformation formed during translation. Only the 0.3 state is observed when poly(Phe) synthesis is prevented by omission of EF-Tu, or in studies on vacant ribosomes. These results suggest that the 0.6 state results from the conformational lability of unlocked ribosomes formed during translocation. An idling state, possibly pertinent to regulation of protein synthesis, is detected in some ribosomes in the poly(Phe) system.

RNA molecules in monovalent salt solutions generally adopt a set of partially folded conformations containing only secondary structure, the intermediate or I state. Addition of Mg2+ strongly stabilizes the native tertiary structure (N state) relative to the I state. In this paper, a combination of experimental and computational approaches is used to estimate the free energy of the interaction of Mg2+ with partially folded I state RNAs and to consider the possibility that Mg2+ favors "compaction" of the I state to a set of conformations with a higher average charge density. A sequence variant with a drastically destabilized tertiary structure was used as a mimic of I state RNA; as measured by small-angle X-ray scattering, it adopted a progressively more compact conformation over a wide Mg2+ concentration range. Average free energies of the interaction of Mg2+ with the I state mimic were obtained by a fluorescence titration method. To interpret these experimental data further, we generated molecular models of the I state and used them in calculations with the nonlinear Poisson-Boltzmann equation to estimate the change in Mg2+-RNA interaction free energy as the average I state dimensions decrease from expanded to compact. The same models were also used to reproduce quantitatively the experimental difference in excess Mg2+ between N and I states. On the basis of these experiments and calculations, I state compaction appears to enhance Mg2+-I state interaction free energies by 10-20%, but this enhancement is at most 5% of the overall Mg2+-associated stabilization free energy for this rRNA fragment.

Our laboratory has previously identified the staphylococcal respiratory response (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. In strain RN4220, SrrAB downregulated production of agr RNAIII, protein A, and TSST-1, particularly under low-oxygen conditions. Work by another group showed that SrrAB regulates energy metabolism genes and indicated that SrrAB may regulate energy transduction in response to changes in oxygen availability. In this study we investigate the role of SrrAB in regulating RNAIII, tst, spa, icaR, and icaA in a clinical isolate of S. aureus, MN8. We employ an inducible antisense vector, pYJY4, in order to repress transcription of srrAB. Transcript levels were assessed by reverse transcription followed by quantitative PCR. Repression of srrAB in rich media under aerobic growth conditions shows that SrrAB is required for expression of tst, spa, and icaR transcripts at wild-type levels. Comparisons made between rich media under aerobic conditions vs low-oxygen conditions show that srrAB transcript levels are not altered by oxygen alone. Previous studies performed on strain RN4220 under low-oxygen conditions indicate that SrrAB represses tst and spa transcript when the amount of oxygen is limited. We propose that, under aerobic conditions, SrrAB enhances the levels of tst, spa, and icaR, while under low-oxygen conditions, SrrAB decreases the levels of these three transcripts.

Colicin E5 specifically cleaves four tRNAs in Escherichia coli that contain the modified nucleotide queuosine (Q) at the wobble position, thereby preventing protein synthesis and ultimately resulting in cell death. Here, the crystal structure of the catalytic domain of colicin E5 (E5-CRD) from E. coli was determined at 1.5 A resolution. Unexpectedly, E5-CRD adopts a core folding with a four-stranded beta-sheet packed against an alpha-helix, seen in the well-studied ribonuclease T1 despite a lack of sequence similarity. Beyond the core catalytic domain, an N-terminal helix, a C-terminal beta-strand and loop, and an extended internal loop constitute an RNA binding cleft. Mutational analysis identified five amino acids that were important for tRNA substrate binding and cleavage by E5-CRD. The structure, together with the mutational study, allows us to propose a model of colicin E5-tRNA interactions, suggesting the molecular basis of tRNA substrate recognition and the mechanism of tRNA cleavage by colicin E5.

Previous work has demonstrated that Escherichia coli DbpA is a nonprocessive RNA helicase that can disrupt short RNA helices on either the 5' side or 3' side of hairpin 92 of 23S rRNA. Here the directionality of the helicase activity of DbpA was determined by using substrates containing a short reporter helix in the presence of a second adjacent helix of varying stability placed either 5' or 3' of the reporter helix. When the second helix was on the 5' side of the reporter helix, it had no effect on the dissociation rate of the reporter helix. However, when the second helix was on the 3' side of the reporter helix, its dissociation rate determined the dissociation rate of the reporter helix. This defines DbpA as a 3' --> 5' helicase. Like other helicases, DbpA requires a single-stranded RNA loading site on the 3' side of the duplex for disruption to be observed. Since the loading site could be on either strand of the helix that was disrupted, hairpin 92 does not influence the directionality of the helicase but only aids in targeting RNA substrates.

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.