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Cell Reports, Volume 3, Issue 2, 497-508, 14 February 2013
The Impact of Aminoglycosides on the Dynamics of Translation Elongation
Albert Tsai1, 3, Sotaro Uemura1, 4, Magnus Johansson1, 5, Elisabetta Viani Puglisi1, R. Andrew Marshall1, 6, Colin Echeverría Aitken1, 7, Jonas Korlach8, Måns Ehrenberg5 and Joseph D. Puglisi1, 2, ,
1 Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA
2 Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, CA 94305-5126, USA
3 Department of Applied Physics, Stanford University, Stanford, CA 94305-4090, USA
4 Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan
5 Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, S-751 24 Uppsala, Sweden
6 McKinsey & Company–Silicon Valley, 3705A Hansen Way, Palo Alto, CA 94304, USA
7 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA
8 Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA
Graphical Abstract
► Combined single-molecule, structural, and biochemical studies on aminoglycoside action ► Aminoglycosides disrupt translation through inhibiting the dynamics of elongation ► Apramycin blocks translocation in translation elongation ► Paromomycin and gentamicin disrupt ribosomal rotation and translocation
- Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition.
The ribosome is the molecular machine that rapidly and accurately interprets the genetic code on the messenger RNA (mRNA) to synthesize proteins (Green and Noller, 1997). During elongation, the ribosome repeats the cycle of selecting a transfer RNA (tRNA) molecule matching the codon in the 30S A site, incorporating the amino acid from the selected A-site tRNA into the polypeptide on the P-site tRNA, translocating the A- and P-site tRNAs to the P and E sites, and stepping precisely three bases in the 3′ direction (Korostelev et al., 2008; Wintermeyer et al., 2004; Zaher and Green, 2009). To elongate with an optimal balance of speed and accuracy, the ribosome employs G protein elongation factors (elongation factor thermo unstable [EF-Tu] and elongation factor G [EF-G] in bacteria) to facilitate key steps during the process (Nilsson and Nissen, 2005). Using the energy from guanosine triphosphate (GTP) hydrolysis, EF-Tu enhances the rate and specificity of tRNA selection and EF-G catalyzes translocation.
The central role of translation makes the ribosome a rich target for clinically important small-molecule antibiotics. They employ diverse strategies to interfere with translation and fall into several distinct classes, including macrolides, tetracyclines, and aminoglycosides (Benveniste and Davies, 1973; Böttger, 2006; Davies et al., 1965; Perzynski et al., 1979; Woodcock et al., 1991). Biochemical and structural studies of these antibiotics have shed light on their mechanism and have, in turn, provided clues to the molecular workings of the ribosome and its ligands (Yonath, 2005). Here, we focus on the clinically important aminoglycosides as a representative group of translational inhibitors.
Aminoglycosides contain a central deoxystreptamine ring with amino-sugar modifications (4,5 and 4,6 disubstituted deoxystreptamine) and include neomycin, paromomycin, gentamicin, kanamycin, and the fused-ring compound, apramycin. Aminoglycosides disrupt the fidelity of tRNA selection and block translocation (Benveniste and Davies, 1973; Davies and Davis, 1968; Davies et al., 1965). Nuclear magnetic resonance (NMR) and X-ray crystal structures (Fourmy et al., 1998) as well as biochemical and molecular biological studies (Böttger et al., 2001; Powers and Noller, 1991) have revealed the structural basis for specific aminoglycoside binding to bacterial ribosomes. Aminoglycosides bind in the major groove of the 16S ribosomal RNA (rRNA) decoding site, forming specific contacts with conserved nucleotides G1494 and U1495; ring I of the aminoglycosides fits into a prokaryote-specific binding pocket formed by universally conserved nucleotides A1492 and A1493 and the prokaryotic-specific nucleotide A1408, imparting their specificity (Böttger et al., 2001; Recht et al., 1999).
These structural studies suggested a mechanism for aminoglycoside action (Figure 1A). In the presence of a cognate codon-anticodon complex, A1492 and A1493 (Moazed and Noller, 1990) make shape-specific contacts in the minor groove of the codon-anticodon A-form helix (Fourmy et al., 1998; Ogle et al., 2001; Yoshizawa et al., 1999). These contacts assist in distinguishing between correct and incorrect codon-anticodon pairings, as shown by both structural and kinetic investigations. In the absence of tRNA or drug, A1492/A1493 are stacked within an asymmetric internal loop at the base of Helix 44 (h44). Upon codon-anticodon interaction, A1492/A1493 are displaced from h44 to make the contacts discussed above. Binding of either 4,5 or 4,6 disubstituted aminoglycosides to the decoding site mimics this conformational effect, displacing A1492 and A1493 toward the minor groove.
Aminoglycosides alter the kinetics of tRNA accommodation, supporting these structural predictions (Pape et al., 1998, Pape et al., 2000). Paromomycin significantly reduces the dissociation rate of near-cognate tRNA and increases the rate of GTPase activation on EF-Tu by an order of magnitude. These results suggest that aminoglycosides stabilize near-cognate tRNA binding to the ribosome with a 16S rRNA conformation that mimics cognate codon-anticodon recognition. While the structural basis of aminoglycoside-induced miscoding has become clear, little is known about how they inhibit the subsequent steps of elongation. The relative importance of miscoding and inhibiting translocation to the potency of the drugs has not been determined. Here, we use a combined dynamics and structural approach to understand how distinct aminoglycosides disrupt translation.
We probed the mechanism of translational inhibition by an aminoglycoside from each of the three distinct structural classes: apramycin (fused-ring), paromomycin (4,5-linked), and gentamicin (4,6-linked) (Figures 1B–1D) using single-molecule, structural, and biochemical approaches. We solved the solution structure of apramycin bound to the bacterial decoding site by NMR spectroscopy, providing a structure to the repertoire of drug-RNA complexes. We determined the effects of all three drugs on the dynamics of translation using an array of single-molecule techniques (Aitken et al., 2010): we monitored the global conformation of the ribosome during elongation (Aitken and Puglisi, 2010) and the dynamics of tRNA accommodation by fluorescence resonance energy transfer (FRET) (Blanchard et al., 2004a) and used zero-mode waveguides (ZMW) to track multiple tRNAs through several elongation cycles at near physiological concentrations (Levene et al., 2003; Uemura et al., 2010). We also used bulk kinetics (Johansson et al., 2011) to measure the effects of aminoglycosides on rates of peptide bond formation and subsequent translocation. This combination of single-molecule dynamics, structural, and biochemical techniques reveals the mechanism of aminoglycoside action at an unparalleled level of detail.
Apramycin, Paromomycin, and Gentamicin Target Distinct Aspects of the Elongation Cycle
To monitor the global effects of aminoglycosides on elongation, we used intersubunit FRET between site-specifically labeled Escherichia coli 30S and 50S subunits (Aitken and Puglisi, 2010; Dorywalska et al., 2005; Marshall et al., 2008) that enable real-time monitoring of single ribosomes performing multiple elongation cycles. For each cycle of elongation, the ribosome rotates the 30S body relative to the 50S and then reverses the rotation, controlled respectively by peptide bond formation upon tRNA accommodation and translocation. The nonrotated state is detected by high FRET and the rotated state by a lower FRET value. The resultant FRET cycles report on the processivity and dynamics of the ribosome at each codon (Figures 2A and 2B) (Aitken and Puglisi, 2010). All three aminoglycosides significantly inhibited elongation efficiency (Figures S1A–S1C; see Extended Results for details). To probe the mechanistic origins of translation inhibition by each aminoglycoside, we leveraged the ability of this single-molecule FRET approach to detect codon-specific effects on the dynamics of 30S body rotation and counterrotation during elongation (Figures 2C–2F and S2A–S2D) (Aitken and Puglisi, 2010; Marshall et al., 2008).
At 1 μM, apramycin did not measurably affect the lifetime (∼4 s) of the high-FRET, or nonrotated state, where the ribosome selects tRNA and performs peptidyl transfer. In contrast, apramycin lengthened the low FRET, or rotated state, where the ribosome waits for EF-G to drive translocation by 2-fold from 5 s without drugs to ∼10 s. Increasing apramycin concentration to 10 μM further increased the rotated ribosome lifetime to 3-fold (∼15 s) longer than minus drugs, but did not otherwise introduce any additional effects on elongation. Increasing the EF-G concentration to 160 nM (from 80 nM, Figures S2E–S2G) partially rescued the rotated ribosome lifetime (1.6-fold longer than that minus drugs [∼8 s]).
At 1 μM paromomycin, the nonrotated state lifetime of the ribosome at the first codon was increased 2-fold (∼10 s), but was unaffected at subsequent codons. Paromomycin also lengthened the rotated state lifetime to a lesser extent by 1.4-fold (∼7 s). Increasing the concentration to 10 μM further lengthened the first nonrotated state lifetime to 3-fold (∼15 s) and the rotated state lifetimes to 2-fold of minus drugs (∼10 s). Additionally, paromomycin lengthened the rotated state specifically over the first codon to 4-fold (∼20 s) from normal. However, this effect is not observed at 1 μM, suggesting a possible alternative binding site (Borovinskaya et al., 2007). Overall, paromomycin displays a more complex set of effects on the elongation cycle and appears to participate in unique interactions with the ribosome at the first codon of the mRNA.
At 1 μM, gentamicin had the most severe effects on the nonrotated state lifetime, lengthening it by 3-fold (∼15 s) across all codons, suggesting a mechanism distinct from paromomycin. However, similar to paromomycin, gentamicin slightly lengthened the rotated lifetime of the ribosome by 1.6-fold (∼8 s). Addition of 10 μM gentamicin led to no further increase in the nonrotated ribosome lifetime; however, similar to paromomycin, a secondary effect of a lengthened rotated ribosome lifetime (to ∼14 s, a 2.8-fold increase over no drugs) over the first codon was observed.
In addition to impacting different phases of elongation, the three aminoglycosides also induce different levels of miscoding, as described in the Extended Results (Figures S1D–S1F).
Following Real-Time tRNA Transit and Sampling in the Presence of Aminoglycosides Using ZMW
We next probed the dynamics of aminoglycoside action from the perspective of tRNA binding by directly tracking in real-time labeled tRNA binding and transit through the ribosome in zero mode waveguides (ZMW). The sequence, frequency, and length of each tRNA signal report on the fidelity of tRNA selection and on the speed of elongation was based on the dwell time of the tRNA (Figures 3A and 3B). We have used this approach previously to track ribosomal compositional dynamics during initiation and elongation (Tsai et al., 2012; Uemura et al., 2010).
In the absence of aminoglycosides, stable binding and transit of multiple tRNAs on the ribosome were observed as regular pulse cycles of repeating Cy5 followed by Cy2 signals with regular times between pulses of 10∼20 s (Figures 3C, 3D, and S3A–S3C), as observed previously (Uemura et al., 2010). In the presence of 10 μM apramycin, the time between pulses lengthened significantly to 30∼40 s, a nearly 2-fold increase, where the pulse sequence was still recognizable. Addition of 10 μM paromomycin and gentamicin introduces frequent metastable tRNA binding events of the length of 1 ∼ 5 s, and the sequences of those events do not match the codon sequence of the 6(FK) mRNA used for the experiments (Figure 3E). The abundance of these short-lived events complicates the identification of binding events, resulting in accommodation and translocation. See the Extended Results for the detailed analysis on how the aminoglycosides alter tRNA binding behavior (Figures S3D–S3F). This result is consistent with our intersubunit FRET experiments that both aminoglycosides severely impact tRNA selection.
Following Real-Time tRNA Dynamics in the Presence of Aminoglycosides
To delineate the distinct effects of aminoglycosides on tRNA sampling and selection, we followed FRET between a P-site fMet-(Cy3)tRNAfMet and an incoming A-site Phe-(Cy5)tRNAPhe in the presence of antibiotics for an mRNA coding for Met-Phe-Lys (MFK). The evolution of this FRET signal reports on the decoding and accommodation of the A-site tRNA as well as classical to hybrid state fluctuations of the tRNA after peptide bond formation (Blanchard et al., 2004b; Figures 4 and S4A–S4C).
Apramycin did not significantly alter the dynamics of tRNA compared to no drugs. In both cases, FRET events proceeded to a high (0.8) FRET state within 100 ms of the appearance of FRET and subsequently fluctuated between high (0.8) and medium (0.5) FRET, consistent with rapid tRNA selection and accommodation and peptide bond formation followed by classical–hybrid dynamics (Blanchard et al., 2004b). However, apramycin increased the overall tRNA FRET lifetime from 10 s in the absence of drugs to 18 s (Document S1. Tables S1 and S2). By blocking of translocation, apramycin could prevent the eventual dissociation of tRNAfMet from the E site, preserving FRET.
In contrast, paromomycin significantly altered the behavior of tRNA on the ribosome and induces many short-lived excursions (<1 s) from zero FRET to a high (>0.7) FRET level consistent with accommodated tRNA (Figure S4F). These events dropped the total FRET lifetime to 1.7 s. While these events are too long to be tRNA sampling attempts (<30 ms), they are much shorter than normal tRNA accommodation without drugs. Longer-lived (1∼5 s) high-FRET events with little to no classical–hybrid state fluctuations were also observed (Figure S4G), consistent with single-molecule FRET measurements by Blanchard and coworkers (Feldman et al., 2010). Together with our ZMW measurements, these data suggest that paromomycin stabilizes tRNAs when they sample the A site and shifts the classical–hybrid tRNA conformation equilibrium strongly toward the classical state. The addition of EF-G enriched longer-lived events, as described in the Extended Results (Figure S4C).
Gentamicin induced behavior similar to that of paromomycin in tRNA-tRNA FRET experiments, with short (<1 s) high-FRET events suggesting that the A-site tRNA has proceeded nearly to accommodation. A longer-lived high-FRET population with limited exchanges between the classical and hybrid tRNA conformations was also observed. These high-FRET events are longer lived than those observed in paromomycin, with a lifetime approaching the photobleaching time of the FRET pair (18 s). The overall fraction of the short-lived population is also lower than paromomycin, as shown in the higher relative density of the long events in the postsynchronized plot and the longer total tRNA lifetime (6.8 s). These differences suggest a mechanism distinct from paromomycin.
Paromomycin Induces Miscoding
To probe how paromomycin alters tRNA selection fidelity, we repeated our tRNA-tRNA FRET experiments with an mRNA encoding Met-Leu-Lys (MLK), in which the Leu codon is CUU, near-cognate to the Phe UUU codon. Delivery of Phe-(Cy5)tRNAPhe to immobilized ribosomes initiated with fMet-(Cy3)tRNAfMet in the P site tested the ability of the ribosome to distinguish between cognate and near-cognate tRNA in the A site. Without drugs and in the presence of apramycin, few detectable tRNA FRET events were observed. Thus, the ribosome was able to discriminate against the near-cognate tRNA; mRNA-independent binding events did not produce FRET and so were not detected in these experiments (Blanchard et al., 2004a).
In the presence of paromomycin, we observed many detectable tRNA FRET events on the near-cognate CUU codon (Figures S4D and S4E). Similar to the cognate mRNA experiment, we observed short excursions to high FRET as well as longer-lived (1∼5 s) high-FRET events that showed very limited classical/hybrid fluctuations. The overall lifetime of tRNA FRET in these experiments (1.4∼1.9 s) was unchanged as compared to tRNA FRET lifetimes on the cognate MFK mRNA with paromomycin. In contrast to the MFK cognate experiment, about 10% of longer-lived events (1∼3 s) were locked in a medium-FRET state (∼0.5), consistent with progression to EF-Tu GTPase activation but not full A-site accommodation (Lee et al., 2007). Pape and coworkers previously observed reduced rates of EF-Tu conformational change and tRNA accommodation after GTPase activation on a near-cognate codon by the addition of paromomycin (Pape et al., 2000). Overall, however, the behavior of tRNA on the cognate MFK and near-cognate MLK mRNAs was remarkably similar in the presence of paromomycin. These results further support the view that paromomycin interferes with the ribosome in distinguishing between cognate and near-cognate tRNA.
The Apramycin Binding Site on the Ribosome Is Distinct from that of Other Aminoglycosides
To explore the structural origins of apramycin function, we determined the NMR solution structure of apramycin in complex with a 27-nucleotide RNA duplex that mimics the A site of the ribosome and the primary binding site of the aminoglycoside antibiotics (Recht et al., 1996). The NMR structure of paromomycin and gentamicin C with this model A-site oligonucleotide were previously published (Fourmy et al., 1996, Fourmy et al., 1998; Yoshizawa et al., 1998), and crystal structures with both oligonucleotide and ribosomal particles are available (Carter et al., 2000; François et al., 2005). Data on the RNA-apramycin complex were acquired on both unlabeled and uniformly 13C, 15N-labeled RNAs (Lukavsky et al., 2003; Lukavsky and Puglisi, 2005). A total of 623 nuclear Overhauser effect (NOE) distance restraints, including 32 drug-RNA NOEs, 111 dihedral restraints, and 23 residual dipolar coupling restraints were used in the structure calculations. The conformation of the drug-RNA interface was well defined by the NMR data: the root-mean-square deviation of the core RNA (U1406-U1410; A1490-U1495) and apramycin for the final 32 lowest energy structures was 1.21 Å (PDB ID 2M4Q).
As with all previous aminoglycoside-RNA complexes, including paromomycin and gentamicin C1a, apramycin binding to the A site was guided by interaction of the 2-deoxystreptamine ring with G1494 and U1495 (Figures 5 and S5) through hydrogen bonding interactions with N7 position of G1494 and the O4 position of U1495. However, the rigid structure of apramycin led to a distinct binding mode compared to that in the NMR structures of paromomycin- and gentamicin-RNA (Fourmy et al., 1996; Yoshizawa et al., 1998). Apramycin did not penetrate deeply in to the aminoglycoside binding pocket formed by A1408, A1492, and A1493, instead making stabilizing interactions with the phosphate backbone. As a result, apramycin did not stabilize the extrahelical, or destacked, conformation of A1492 and A1493 observed in the presence of paromomycin and other miscoding aminoglycosides. Apramycin binding to the oligonucleotide in solution favors the intrahelical, or stacked, conformation of the A1408, A1492, and A1493 observed in the structure of the free A-site oligonucleotide (Recht et al., 1996). Hygromycin B, another structurally unique, nonarchetypal antibiotic, binds the A site but does not cause destacking of A1492 and A1493; hygromycin B inhibits translocation without causing significant miscoding (Brodersen et al., 2000; Cabañas et al., 1978).
Effects of Apramycin, Paromomycin, and Gentamicin on Peptide Bond Formation Rates
The intersubunit FRET experiments show that paromomycin and gentamicin inhibit 30S body rotation during elongation. This could result from delays in peptide bond formation due to disruption in tRNA selection or steps subsequent to peptide bond formation. Accordingly, we assayed the effect of the three aminoglycosides on peptide bond formation kinetics in bulk experiments, as previously described (Johansson et al., 2011). The rates of GTP hydrolysis (kGTP) and dipeptide formation (kpep) for all three drugs remained unchanged compared to no drugs (Figure 6). As a control, we also measured the rate of formation of tripeptide (fMet-Phe-Phe) in each case (Figure S6). The rate of disappearance of f[3H]Met, reflecting the rate of dipeptide formation, was also the same in each case. The rate of formation of tripeptides was, however, significantly decreased by the presence of each one of the drugs, in line with all three drugs slowing down the ribosome in elongation. These results suggest that the fraction of drug-bound ribosomes was close to 100% in the experiments.
Apramycin, Paromomycin, and Gentamicin Efficiently Inhibit Translation Elongation
Using the intersubunit FRET signal, we assayed the effects of apramycin, paromomycin, and gentamicin on the processivity of individual ribosomes at 80 nM tRNA and 80 nM EF-G. The number of elongation (high-low-high FRET) cycles that each ribosome performed on our 12-codon mRNA is plotted as histograms. Without drugs, many ribosomes translated the entire mRNA with the distribution showing a clear peak at codon 12 (Figure S1A). However, in the presence of 1μM aminoglycosides (Figure S1B), this peak – representing ribosomes whose translation is limited by mRNA length – disappears, and is replaced by peaks at codons 2∼4 for apramycin, 1∼3 for paromomycin, and 1 for gentamicin, the strongest effect observed. Similar levels of inhibition were observed at 10 μM drug concentration (Figure S1C), suggesting that 1 μM is at or above saturating concentration for these effects.
Paromomycin and Gentamicin Induce Misincorporation of tRNAs
To test whether apramycin, paromomycin, and gentamicin promote misincorporation and translocation through near-cognate codons, we followed the intersubunit FRET signal of ribosomes translating an mRNA in which the coding region of three Phe codons (UUU) is followed by a stop codon (UAA), which is near-cognate to Lys (AAA), and then four more Phe codons (UUU). In the presence of Phe-tRNAPhe and Lys-tRNALys, ribosomes that do not misincorporate the near-cognate Lys-tRNALys should perform three rounds of elongation; the fraction of ribosomes that performs more than three rounds of elongation reports on the degree of miscoding in this stop codon readthrough assay within our observation timeframe (Figures S1D–S1F). Due to ribosome slippage on poly-U mRNAs and differences in the compositions of translation systems between different studies, the miscoding frequencies reported are relative and not interpreted as absolute miscoding rates.
In the absence of aminoglycosides, this miscoding frequency is 2.5%, establishing the baseline of translational fidelity for our assay. In 10 μM apramycin the miscoding frequency triples to 7.5%. Both paromomycin and gentamicin, however, increase this miscoding frequency by an order of magnitude to 30%. Even with only Phe-tRNAPhe (non-cognate to the stop codon), paromomycin still induced 6-fold more misincorporation on 15% of ribosomes. These data suggest that paromomycin and gentamicin severely disrupt, whereas apramycin exerts a weaker effect on tRNA selection.
Analysis of ZMW Experiments with Paromomycin and Gentamicin
The lifetimes of tRNA binding events in the presence of aminoglycosides confirm hindered translation elongation during and after tRNA selection. When all visible events were assigned (Figures S3D–S3F), the lifetimes of tRNA events are consistently longer for apramycin compared to no aminoglycosides for Phe-(Cy5)tRNAPhe by 3.5-fold (7.6 s versus 2.2 s without drugs) and Lys-(Cy2)tRNALys by 1.7-fold (6.9 s versus 4.1 s without drugs). Compared to no drugs, the frequencies of tRNA events in apramycin are lower for tRNAPhe by 2-fold (0.027 s-1 versus 0.054 s-1 without drugs) or similar for tRNALys (0.031 s-1 versus 0.035 s-1 without drugs). On average, in apramycin, tRNAPhe events occupy 0.21 and tRNALys events occupy 0.24 of each trace, approximately 2-fold increases from 0.12 and 0.13 for no aminoglycosides respectively (an occupancy of 1 means that the entire trace is covered by 1 tRNA event). The longer lifetimes and, thus, higher occupancy of tRNA events that apramycin induces are consistent with our intersubunit FRET result that apramycin blocks the EF-G dependent translocation step, which leads to longer tRNA transit times.
The presence of paromomycin affected only the event frequencies, but not the lifetime, consistent with its stronger effect on the tRNA selection step compared to translocation. The overall tRNA lifetimes for both tRNAPhe (3.7 s) and tRNALys (4.0 s) were not affected by paromomycin, whereas visible event frequencies increased by 1.5-fold (0.082 s-1) for tRNAPhe and 2.9-fold (0.10 s-1) for tRNALys. Accordingly, the overall occupancy of tRNA events increased significantly by 2.5 times (0.30) for tRNAPhe and 3.2 times (0.41) for tRNALys. Paromomycin significantly increased the number of visible tRNA events (>100 ms, the limit of reliably assigning an event), consistent with the structural prediction that paromomycin stabilizes tRNAs in the A site. In the absence of drug, sampling events are < 30 ms and should be rarely observed in 30 frames per second ZMW experiments. Finally, tRNA lifetimes with gentamicin are either longer with tRNAPhe by 3.6-fold (8.0 s) or similar to no drugs with tRNALys (3.9 s), which could be the result of many ribosomes stalling over the first Phe codon. The tRNA frequencies in gentamicin either remained similar to no drugs for tRNAPhe (0.058 s-1) or increased for tRNALys by 2.3-fold (0.080 s-1). Overall, the changes in event frequencies were not as significant as for paromomycin. The overall event occupancy for gentamicin is also high at 3.8 times from normal (0.46) for tRNAPhe and 2.4 times (0.31) for tRNALys. Gentamicin and paromomycin both act during the tRNA binding and selection stage, with distinct nuances as observed in the other assays.
tRNA-tRNA FRET Dynamics in the Presence of EF-G
In experiments without aminoglycosides and with apramycin, adding 500 nM EF-G did not noticeably changed the evolution of the FRET signal upon tRNA binding to the A site. FRET events proceeded to a high (0.8) FRET state within 100 ms of the appearance of FRET and subsequently fluctuated between high (0.8) and medium (0.5) FRET, consistent with rapid tRNA selection and accommodation, and peptide bond formation followed by classical-hybrid dynamics.
With paromomycin, the presence of 500 nM EF-G enriched moderately long-lived (1∼5 s) high FRET (>0.7) events, as shown in the postsynchronized plot, perhaps as a result of EF-G trapping the A-site tRNA. As short tRNA events still dominated, the total FRET lifetime was similarly short at 1.8 s when compared to no EF-G.
Aminoglycosides are thought to affect translation by decreasing the fidelity of tRNA selection and blocking translocation. We employed a battery of complementary single-molecule methods, supported by structural and biochemical techniques, to form a coherent picture of the mechanism of action for three structurally distinct antibiotics: paromomycin, gentamicin C, and apramycin (Figure 7).
Apramycin Slows Translation via One Primary Inhibition Mechanism
Apramycin has the clearest but the most atypical mode of action. The NMR solution structure of the apramycin-ribosomal RNA complex suggested a distinct structural mechanism for inhibition of translocation. The apramycin structure shows that the drug binds in the major groove of the 16S rRNA decoding site similar to other aminoglycosides. However, the drug does not induce the destacked conformation of A1492/A1493, as observed during correct codon-anticodon pairing. This differs with X-ray crystal structures showing A1492/A1493 destacked for all aminoglycosides (see Extended Discussion). However, the recent crystal structure of apramycin bound to the 30S subunit-tRNA-mRNA complex suggests that the drug would block unstacking of A1492 (Matt et al., 2012), consistent with this interpretation. The bias for a stacked conformation of A1492/A1493 in the presence of apramycin is supported by lower level of miscoding observed here and by others (Matt et al., 2012). This bias could additionally reduce the impact of apramycin on elongation dynamics over cognate codons prior to 30S body rotation, as evidenced by the normal nonrotated lifetime in intersubunit FRET, behavior in tRNA-tRNA FRET that is indistinguishable from no drugs, low tRNA sampling frequency in the ZMW (see Extended Discussion), and lack of effect on cognate kpep.
Apramycin mainly blocks translocation; it significantly lengthens the rotated state lifetime, where the 70S complex is waiting for EF-G in order to translocate. While a previous single-molecule work (Feldman et al., 2010) has correlated inhibition in translocation to perturbations in tRNA conformational dynamics, apramycin breaks this trend, as it strongly inhibits translocation without inducing any noticeable increase in the occupancy of the classical tRNA conformational state in our tRNA-tRNA FRET experiment. Apramycin may instead lock the A site in an unfavorable conformation for EF-G, effectively increasing the energy barrier that EF-G must overcome to translocate. Similar effects have been observed for the translocation inhibitor spectinomycin, which binds the 30S subunit (Aitken and Puglisi, 2010). Apramycin inhibits the rate of elongation for about ten codons into the message, yet it gradually wears off such that the rotated-state lifetime has mostly recovered by the end of our 6(FK) mRNA. This suggests that the early phase of translation is more vulnerable to antibiotic action.
Paromomycin and Gentamicin Exhibit More Complex Modes of Action
Paromomycin disrupts multiple aspects of elongation, consistent with the conformational changes observed by structural methods. By favoring the destacked A1492/A1493 conformation (Fourmy et al., 1998), it causes severe miscoding in our stop codon readthrough assay. From tRNA-tRNA FRET experiments with a near-cognate codon (MLK) mRNA, the many observable events suggest that paromomycin slows the dissociation of an incorrect tRNA from the A site, consistent with prior kinetic measurements (Pape et al., 2000). Strikingly, paromomycin still retained half of its miscoding effect in our miscoding assay, where only the noncognate tRNAPhe (to the stop codon) was present. As the total tRNA concentration in the experiment is halved, this result suggests tentatively that destacking of A1492/A1493 can induce miscoding with any tRNA available. The incorrect pulse sequence in ZMW experiments with paromomycin further demonstrates that the ribosome has lost its ability to distinguish correct codon-anticodon interactions.
The effects of paromomycin on elongation appear during both global conformational states of the ribosome and sometimes in a codon-specific manner. The frequent short tRNA FRET events indicate that the ribosome goes through many futile cycles where it accepts a tRNA into the A site, bypassing initial selection, but eventually rejects it. The high tRNA FRET level suggests that the A-site tRNA has proceeded beyond GTP hydrolysis by EF-Tu. Normal bulk peptide bond formation rate suggests that some tRNAs proceed as far as transferring the peptide chain from the P-site tRNA to the A-site tRNA. Combined with the increased nonrotated state lifetime in intersubunit FRET experiments, this suggests that paromomycin significantly increases the energy barrier to 30S body rotation, decoupling it from peptide bond formation. If a ribosome stalls early in elongation with a short peptide chain, it could risk losing the A-site tRNA by dissociation. Such ribosomes are left with a deacylated P-site tRNA and are unable to translocate normally, even when a correct tRNA binds in the A site, as indicated by the frequent short-lived binding events observed in the ZMW for stalled traces. Eventually, the ribosome might translocate spontaneously with an acylated A-site tRNA bound and reset itself with an amino acid on the P-site tRNA; however, accumulation of prematurely released peptidyl-tRNA could be toxic. Consistent with that hypothesis, some traces that halted at the first codon in both intersubunit FRET and ZMW experiments subsequently resumed accommodating tRNAs (Figure 3F). These severely malfunctioning ribosomes can also become candidates for termination by release factors in vivo (Dunkle and Cate, 2010), aborting translation after significant energy expenditure.
Paromomycin additionally prolongs the rotated state in general; however, this effect is weaker than apramycin. Paromomycin limits the classical/hybrid tRNA FRET fluctuations, favoring the classical state, which is not conducive for translocation, consistent with observations of Feldman et al., 2010. Alternatively, the stabilized, destacked conformation of A1492 and A1493 may conflict with EF-G function to reset the ribosome (Taylor et al., 2007). For both mechanisms, paromomycin effectively increases the energy barrier for the ribosome to translocate. Additionally, paromomycin can significantly lengthen the rotated lifetime of the first codon, but this is only observed at 10 μM paromomycin concentration. This suggests the presence of a secondary binding site; however, the mechanism for this codon-specific effect is not clear.
Gentamicin C, which also promotes the destacked A1492/A1493 conformation upon binding, induces miscoding as paromomycin. Gentamicin causes a similar increase in rotated ribosome lifetimes as well as locks the tRNAs into the classical conformation in tRNA-tRNA FRET experiments (Feldman et al., 2010). Additionally, gentamicin induces short-lived tRNA-binding events, like paromomycin in ZMW experiments. Unlike paromomycin, gentamicin increases the energy barrier to ribosome rotation across all codons, resulting in more severe disruptions than paromomycin. Gentamicin is 4,6 linked, and its ring III element contacts G1405 in the decoding site (Yoshizawa et al., 1998), whereas the additional ring contacts in 4,5 disubstituted aminoglycosides occur to the helical stem closed by C1409-G1491 (Fourmy et al., 1996). These different contacts and their effects on local and global ribosomal dynamics may explain the different mechanistic observations. Alternatively, secondary binding sites may also contribute. Intriguingly, gentamicin has the strongest inhibitory effect and is the most important of the three drugs in clinical applications.
Dissecting the Complex Effects of Aminoglycosides from Multiple Perspectives
By employing complementary single-molecule techniques, buttressed by structure and biochemical assays, our results provide a comprehensive understanding of aminoglycoside mechanisms. Their mode of action is far from simple and involves significant modifications to the delicate energy landscape of elongation. Aminoglycosides have distinct profiles of elongation inhibition, and their structural diversity indeed translates into a diversity of mechanisms. While aminoglycosides are thought to be effective by inducing miscoding and hindering translocation, we have observed significant differences in the level of miscoding and effects on steps not directly linked to translocation, which for paromomycin and gentamicin were stronger than their effects on translocation. Despite the complexity of their mechanism, a common theme emerges where they significantly increase energy barriers for the ribosome subunits to rotate and to translocate. As the 1.5- to 4-fold inhibitions over each codon to various steps of the elongation cycle accumulate across multiple codons, these drugs can effectively stall the ribosome and cause severe disruptions to elongation. In fact, even with our 12-codon model mRNA, we already see a dramatic decrease in the number of codons translated during our observation timeframe. These observations agree with the original discussions of Kurland (Kurland, 1987) and others that aminoglycosides are bacteriocidal because of their inhibition of elongation efficiency, not miscoding. How the local conformational changes caused by aminoglycoside binding to the 30S decoding site affect the global behavior of the ribosome and how miscoding aminoglycosides modify the balance of tRNA selection fidelity versus efficiency remain open questions (Johansson et al., 2012).
Differences between NMR Solution and X-Ray Crystal Structures
NMR solution structure results contrast with static crystal structures of paromomycin, gentamicin and apramycin bound to a similar RNA oligonucleotide, all of which demonstrate a de-stacked conformation of A1492 and A1493 (Han et al., 2005). The crystal structure of the apramycin-RNA complex would lead to 23 NOE violations (>0.4Å), and predicts 8 strong NOEs not observed in the solution structure. Interestingly, none of the RNA-aminoglycoside solution structures are fully consistent with the exaggerated de-stacked conformation of A1492 and A1493 observed in the X-ray crystal structures. Instead, the NMR solution structures suggest a conformation of A1492 and A1493 that is biased toward the minor groove when rRNA is in complex with paromomycin and gentamicin C1A, and a conformation biased toward being stacked when in complex with apramycin (Lynch et al., 2003). The differences are reconcilable if both conformations existing in solution, albeit with different relative populations depending on the aminoglycoside. The crystal structure could be the conformation that is more conducive to crystal packing and, if the two conformations are in rapid exchange (less than 1 ms time scale, which is likely for a stacking-destacking reaction), the solution structure represents an average of the two populations (Dethoff et al., 2012). The model oligonucleotides do not fully recapitulate the situation within the full ribosome. The conformation of the apramycin-30S subunit complex was solved in the context of the codon-anticodon helix interaction in the A site. In addition, ribosomal protein S12 makes contacts to stabilize the stacked out conformation of A1492. Nonetheless, it is clear from the NMR and crystallographic studies that subtle modulations of the conformational equilibrium of A1492/A1493 by aminoglycosides give rise to their different modes of action.
Experimental Procedures
Single-Molecule Reagents and Buffers
Fluorescently labeled 30S (Cy3 or Cy3B) and 50S (Cy5) subunits, wild-type 30S and 50S subunits, translation factors, S1, mRNA, fluorescently labeled tRNA (fMet-[Cy3]tRNAfMet, Phe-[Cy5]tRNAPhe, and Lys-[Cy2]tRNALys), and unlabeled tRNA (fMet-tRNAfMet, Phe-tRNAPhe, and Lys-tRNALys) were prepared as previously described (Blanchard et al., 2004b; Dorywalska et al., 2005; Marshall et al., 2008). All experiments were conducted in a Tris-based polymix buffer consisting of 50 mM Tris-acetate (pH 7.5), 100 mM potassium chloride, 5 mM ammonium acetate, 0.5 mM calcium acetate, 5 mM magnesium acetate, 0.5 mM EDTA, 5 mM putrescine-HCl, and 1 mM spermidine. All experiments also contained 4 mM GTP. The aminoglycosides used in this study were purchased from Sigma as dry powders and dissolved in water to make stock solutions.
The 5′ biotinylated mRNAs for the intersubunit FRET and ZMW experiments contained the 5′ UTR and Shine-Dalgarno sequence of T4 gene 32 followed by either three Phe (UUU) codons (3F) or six alternating Phe (UUC) and Lys (AAA) codons (6(FK)). Both 6(FK) and 3F have a stop codon after the end of the message followed by an additional 4 Phe (UUU) codons (Aitken and Puglisi, 2010). The mRNAs for tRNA-tRNA FRET experiments either contain the full T4 gene 32 sequence up to the 7th translated codon (MFK) or have the second translated codon changed from Phe (UUU) to a Leu (CUU) (MLK). All mRNAs were chemically synthesized by Dharmacon.
NMR Solution Structure of Apramycin in Complex with A-Site Oligonucleotide Mimic
Near-complete assignment of the proton resonances from the apramycin-RNA complex was accomplished using standard multidimensional, homonuclear, and heteronuclear experiments with unlabeled RNA, and uniformly 13C, 15N-labeled RNA samples (Lukavsky et al., 2003; Lukavsky and Puglisi, 2005). A total of 612 nuclear Overhauser effect (NOE) distance restrains were determined from two-dimensional (2D) and three-dimensional (3D) NOE spectroscopy (NOESY) experiments, of which 32 were intermolecular RNA-apramycin distance restraints. Distance restraints were categorized based on peak intensity into four groups with the following interproton distances: strong (1.8–3.5 Å), medium (2.0–4.5 Å), weak (2.2–6.0 Å), and very weak (3.0–7.0 Å). Dihedral restrains, β, ε, χ, were determined from 3D HCP, 3D heteronuclear multiple quantum coherence (HMQC) total correlation spectroscopy (TOCSY), and 2D NOESY experiments, respectively. Residual dipolar coupling restraints from aromatic N–H and C–H bond vectors were determined by comparing J-coupling constants collected from 2D 13C/15N transverse relaxation optimized spectroscopy (TROSY) spectra in the presence and absence of pf1 bacteriophage. Residual dipolar couplings (RDCs) were determined according to the following formula:
Intersubunit FRET Experiments
We mixed 0.25 μM Cy3B-30S, preincubated with stoichiometric S1, 1 μM IF2, 1 μM fMet-tRNAfMet, 1 μM 5′ biotinylated mRNA, and 4 mM GTP to form 30S preinitiation complexes (PICs) in the Tris-based polymix buffer described above to form 30S PICs (Dorywalska et al., 2005). The PIC mixture was incubated at 37°C for 5 min.
Before immobilizing the 30S PICs on a slide surface, we diluted the mixture with the Tris-based polymix buffer with 1 μM IF2 and 4 mM GTP. The PICs were then immobilized on the surface of a biotin-polyethylene glycol (PEG)-derivatized quartz slide via biotin-neutravidin interactions. We then washed excessive unbound material with the Tris-based polymix buffer containing 1 μM IF2, 4 mM GTP, 1 mM Trolox (to stabilize Cy5 photophysics), and an oxygen-scavenging system (2.5 mM 3,4-dihydroxybenzoic acid and 250 nM protocatechuate deoxygenase; Aitken et al., 2008). We delivered 50 nM Cy5-50S, 1 μM IF2, 80 or 160 nM EF-G, 80 nM ternary complexes, and aminoglycosides (where applicable at the indicated concentration) in the Tris-based polymix buffer using a controlled syringe pump to initiate translation. Phe-tRNAPhe and Lys-tRNALys ternary complexes with EF-Tu(GTP) were formed immediately before delivery, as previously described (Marshall et al., 2008).
ZMW Experiments
The preparation for ZMW experiments was the same as intersubunit experiments with the following exceptions. The first incubation mixture contains 0.25 μM unlabeled wild-type 30S, 0.25 μM wild-type 50S, 1 μM fMet-(Cy3)tRNAfMet, 1 μM 5′ biotinylated mRNA, and 4 mM GTP to form 70S initiation complexes (ICs). The 70S ICs are then immobilized using the same procedure as in intersubunit FRET experiments. The delivery mixture contained 1 μM IF2, 200 nM EF-G, 200 nM Phe-(Cy5)tRNAPhe ternary complexes, 200 nM Lys-(Cy2)tRNALys ternary complexes, and 10 μM paromomycin, apramycin, or gentamicin, comparable to intersubunit FRET conditions.
tRNA-tRNA FRET Experiments
The preparation for tRNA-tRNA FRET experiments was the same as ZMW experiments with the following exception. The delivery mixture contains 1 μM IF2, 500 nM EF-G (where applicable), 25 nM Phe-(Cy5)tRNAPhe ternary complexes, and 10 μM aminoglycosides (where applicable).
Bulk Kinetic Experiments
Bulk kinetic experiments were performed at 20°C and pH 7.5 in polymix buffer (95 mM KCl, 5 mM NH4Cl, 5 mM Mg(OAc)2, 0.5 mM CaCl2, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, and 1 mM DTE) complemented with an energy supply and regeneration system consisting of 2 mM (ATP + GTP), 10 mM phosphoenolpyruvate, pyruvate kinase, and myokinase (Johansson et al., 2011). In the experiments for simultaneous monitoring of dipeptide formation and GTP hydrolysis, preinitiated 70S ribosomal complexes, carrying f[3H]Met-tRNAfMet in P site and displaying the Phe codon UUC in A site, were rapidly mixed with preformed Phe-tRNAPhe:EF-Tu:[3H]GTP ternary complex in a quench-flow instrument (RQF-3, KinTek Corp.) to a final concentration of 200 nM. The reactions were quenched with formic acid at different incubation times and the fractions of products, [3H]GDP/([3H]GDP + [3H]GTP) and f[3H]Met-Phe/(f[3H]Met-Phe + f[3H]Met), were estimated by high-performance liquid chromatography (HPLC) as described (Johansson et al., 2011). The rates of GTP hydrolysis and dipeptide formation were measured in the presence (10 μM) and absence of each drug. The rate of GTP hydrolysis, kGTP, including the association of ternary complex to the ribosome, and the rate of tRNA accommodation/peptidyl transfer, kpep, obtained from the inverse of the difference between the dipeptide formation time and GTP hydrolysis time (Johansson et al., 2011) were estimated in drug absence and in the presence of each one of the drugs. Tripeptide formation was performed by rapid mixing and quenching of mixtures similar to those used for dipeptide bond formation, but with EF-G and EF-Ts added to the ternary complex mix and use of unlabeled rather than labeled GTP.
For further details, please refer to Extended Experimental Procedures.
TIRF Instrumentation and Analysis
We performed the intersubunit and tRNA FRET experiments on a prism-based total internal reflection fluorescence microscope as previously described (Aitken and Puglisi, 2010; Marshall et al., 2008). For FRET illumination, we used a diode-pumped solid-state 532 nm at 1 kW cm-2. A Quad-View device (Photometrics) separated fluorescence emissions into four channels and two of the channels were filtered to correspond to emission spectrum of Cy3 or Cy3B and Cy5. The signal was projected onto two quadrants of a 512x512 EMCCD camera (Andor Technology). We collected videos using the MetaMorph software package (Molecular Devices) and extracted fluorescent traces using in-house MatLab (MathWorks) scripts. Intersubunit FRET videos were collected at an exposure time of 50 ms per frame for 4800 frames (240 s) and tRNA-tRNA FRET videos were collected at an exposure time of 25 ms per frame for 3600 frames (90 s). FRET states were assigned using a previously described hidden Markov model based approach (Aitken et al., 2008) and corrected visually if necessary. Lifetimes for the intersubunit FRET experiments were assigned from only productive states (e.g., low FRET states that was followed by a high FRET state and vice versa) to eliminate effects from photophysical effects and molecules trapped in a long-lived non-functional state. Lifetimes for tRNA-tRNA FRET experiments were fitted from all observable non-zero FRET states that do not show abnormal photophysical effects (e.g., blinking and resurrection from long-lived dark state). All lifetimes were fitted to a single-exponential distribution using maximum-likelihood parameter estimation.
ZMW Instrumentation and Analysis
Instrumentation and chips containing 3,000 individual ZMWs were used and prepared as described previously (Uemura et al., 2010) ZMW diameters were in the range of 120-135 nm. Lys-(Cy2)tRNALys, fMet-(Cy3)tRNAfMet and Phe-(Cy5)tRNAPhe fluorescence was detected using direct excitation with lasers at 488, 532 and 632 nm. Laser power for three excitations was 0.5 μW/μm2 for all ZMW experiments. Fluorescence traces were recorded at a rate of 30 frames per second for 5 min. Traces were selected and analyzed using the same set of scripts as TIRF experiments and the tRNA lifetimes were fitted to single exponential distributions using the same method as described for TIRF.
Supported by NIH grant GM51266 (to J.D.P.), the Japan Science and Technology Agency (to S.U.), the Swedish Research Council (to M.E.), and the Knut and Alice Wallenberg Foundation (to M.E.). We thank Alexey Petrov (Stanford), Jin Chen (Stanford), and Seán O'Leary (Stanford) for valuable discussions. J.K. is an employee and stock option holder, and J.D.P. is a consultant of Pacific Biosciences, a company commercializing sequencing technologies.
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Publication Information
Received: August 5, 2012
Revised: November 18, 2012
Accepted: January 22, 2013
Published: February 14, 2013


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Les Miserables Celebrity Fans: Lea Michele, Eric Stonestreet
Dec 26, 2012 @ 12:35 pmEveryone rooted for Prisoner 24601 at the movies, including celebrities! After seeing Les Miserables, Glee star Lea Michele—who made her Broadway debut as the young Cosette at the age of nine—Tweeted "Just saw Les Mis.. is was amazing!! As a member of the Les Mis family I thought they did an amazing job and truly honored the show." Modern Family's Eric Stonestreet echoed the sentiments. "Amazing job @SamanthaBarks in Les Miserables! I sang along with u until I was asked to stop by the people around me and then by management." So have you seen it? Don't wait One Day More!
Plus, see Samantha Barks's off-screen style.

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Vietnam Memorial center searches for D.C. vets
Saturday - 7/6/2013, 8:25am  ET
WASHINGTON - As tourists visit the monuments of Washington on this Independence Day weekend, they'll no doubt make a stop at the Vietnam Memorial.
But a project in the works to expand the memorial is stalling without the help it needs.
An underground education center has been in its planning stage since 2003. Organized by the Vietnam Memorial Fund, the center will tell the stories of the 58,000 veterans who lost their lives in Vietnam.
While the photos and stories of veterans from some states are flowing in or completely submitted, the stories of veterans from the District are not.
"We've run into sort of an interesting problem here in Washington, D.C., where we have 272 casualties (represented)," says Jan Scruggs, founder and president of the Vietnam Memorial Fund. "For some reason, we need another 198 to get all the photographs."
Scruggs is unsure of why Washington has not come through with the photos of its Vietnam vets, though he says it could have to do with the transient nature of the city 40 years ago.
In other states, a group or organization has take on the task of tracking down each veteran, but no one has come forward to do so in D.C.
"Hopefully we'll find somebody in D.C who will start working on this and make a good success out of it - maybe somebody who is interested in the history of Washington, D.C. - because these photographs are important," Scruggs says.
The power of the education center comes from telling the soldiers' personal stories, which are essential to the completion of the museum, Scruggs says.
"These photographs also tell the story, in part, of African-American experience in Vietnam and their courage," he says. "This is a part of America's story and it needs to be told."
People can mail in photos and notes about their Vietnam War veteran, or submit photos online here. To help with the education center, click here.
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Weather and road conditions in California can change quickly. Check out the road conditions reports and live highway cams before your next trip to Northstar California Resort here!
Traveler Information Provided by the California Department of Transportation (CalTrans)
HWY-267 at Northstar Drive |
HWY-267 at SR-28 (Kings Beach) |
I-80 at HWY-267 |
I-80 at SR-89 |
SR-89 at SR-28 (Fanny Bridge) |
HWY-50 at Ski Run Boulevard (South Lake Tahoe) |
HWY-50 at SR-89 (Mays Junction, South Lake Tahoe) |
HWY-50 / SR-89 at Luthar Pass Road (Meyers) |


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Reunion Ride Board
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Use this ride board to connect with other alumni. Read listings below, or post a new announcement.
Contact the alumna/us directly if you see something that matches your plans. Check back frequently for new listings.
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Go Minneapolis is your home for exclusive experiences and deals in Minneapolis. Find all the best Minneapolis coupons here and save on attractions, restaurants and hotels in Minneapolis.
When you come to Minneapolis, we like to make sure that your event goes as smoothly as possible. Our talented staff has the means to provide you with housing, registration and online services, as well as a comprehensive list of on-site services. If you can't find what you're looking for, don't hesitate to check out our staff list. Plus, you can see what other meeting professionals had to say about us, as well as the awards we've won for our work. So rest assured that when you schedule your meeting in Minneapolis you're getting the best.


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If you do not get warm easily in your sleep this is a very nice high quality mattress protector/pad- however my boyfriend and I both are warmer natured and even if I sleep on it alone with the temperature at 68, I will end up sweating. Not sure if it is because of the waterproofing but I'm sad to say we are having to give this one away. Otherwise a very nice comfortable and padded protector.
This is a really nice mattress pad. It is waterproof, but no eplastic is used. It is high loft and very soft. It definitely improves any mattress.
Received this fine quality mattress pad and was very happy. My man and I have slept on this goodness for over a week now and have woken up every night in a pool of our own sweat. All attempts to rectify the situation by turning down the heat, wearing less to bed, etc have ended up in the same result. So, sadly, I think we have to kick this over to the guest bedroom and make our guests suffer. Perhaps too waterproof?
I started my search for a mattress pad and thought nothing but a feather down mattress pad would give the the comfort I was looking for....but given the reviews of many other EXTREMELY expensive mattress pads, I opted for the the Sleep Philisophy Harmony Mattress Pad and am SO HAPPY with my purchase. There is a noticeable difference in the comfort of my bed now -- light and fluffy...like sleeping on air. I have a King Sized Sleep Number Bed -- the pad fits snug and perfect! The only smell I noticed was when I initially took it out of the plastic bad (I blame the bag, not the mattress). That smell dissapated soon after removing it from the bag, so no worries there. I haven't washed it yet, but I expect it will wash fine as the material appear to be good quality. I highly recomend.
I was looking for a mattress pad that not only was waterproof, but also added a little extra cushion to my worn-in mattress. This pad does both & I love it. It even goes through the washer & dryer fantastically too! :) Definitely worth it!
I have owned two of these mattress pads for a few months. They are used for my son's bed. My son is totally disabled and bedridden, so these pads get a lot of use. The pads have consistently come through the wash well. They stay fluffy and continue to protect the mattress from any wetness or stains. They both still look brand new. I'm 100% pleased!
I purchased this mattress pad to go over a new king size mattress. Out of the packaging, the mattress pad was very impressive. Very soft, and great padding. I'm usually very picky with my mattress pads, and I was very pleased with this one....that is until I actually slept on it. I woke up in the middle of the first night thinking I had a fever. I'm not sure what it is about this pad that makes you feel like you're actually cooking in your own bed, but it's very uncomfortable and disrupts sleep. If you're cold-natured, live in very cold climates, have poor circulation, etc, this is probably the pad for you. It's very soft and lovely, but too hot for me.
Actually waterproof and not merely water 'resistant'. Nothing gets trough to mattress. Have older pet with an occasional bladder control problem so product works well for this. Washes and machine dry like a dream.
The mattress pad is very soft and comfortable. It also resists liquid very well. It does exactly what it promises.
We bought this as a waterproof mattress pad for my son's bed. It is very soft and nice quality and actually adds to the comfort of his bed. The only downfall to this mattress pad is that we have found it can not be dried in the dryer or it melts the waterproof backing on the underneath. We plan to buy a second one of the mattress covers so that we have a back up when my son has an accident and it needs to be washed. We just don't have time to wait for it to be washed and air dryed before naptime. I would recommend this cover as we will be purchasing a second one!
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Friends, city mourn Bellaire officer killed on duty
Police veteran, killed in traffic stop that turned deadly, remembered as someone always eager to help others
Melissa Phillip, Houston Chronicle
December 29, 2012 | Updated: December 30, 2012 1:32pm
More than 1,000 people stood Saturday afternoon as the family of slain Bellaire police officer Sgt. Jimmie Norman walked into Houston's First Baptist Church to take their seats.To continue reading this story, you will need to be a digital subscriber to HoustonChronicle.com.
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Lets Go!
To reserve a seat:
- E-mail the Transfer Center at email@example.com
(Include your name, student ID, phone number, email address, and the tour you will be attending.) - Or call the Transfer Center at (949) 582-4328
- Or visit the Transfer Center
Sign up early, as the tours fill up quickly. Transportation provided by the Transfer Center.
Day | Date | Campus | Time |
Check | Back | This | Spring! |


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The tree is up? The lights are on? he's still dragging the tree on my screen.
The tree is up? The lights are on? he's still dragging the tree on my screen.
Easy for you to say....you have a keyboard. (I think I know had to figure it out on my settings.)
I think it's amazing how animated Jake's been decorating the tree. Real cool and original!
Lol. That's cute.
He is a busy lil crow. I love it!
The primary difference as I see it is that the Mac OS is easier to use. Used to be it was more stable, meaning fewer crashes, freezes, reboots and reinstalls, than Windows, but I dunno if that's still true. Macs are easier, imo, because they're easier to figure out and they require fewer steps to get the job done, a fact which applies to all aspects of personal computing. I think the addage "Once you go Mac you never go back" is probably close to statistically accurate.


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County Roscommon Vacation Rentals, Villas, & More
Sort rentals by:
Bedrooms & Guests
Luxurious 4 Bedroom House in the West of Ireland
- Bedrooms:
- 4
- Baths:
- 3
- Sleeps:
- 8
from$116/ night
Amenities included
- Linens Provided
- Children's Toys
- Television
- Microwave
- Clothes Dryer
- Parking
- Garden
- Dish Washer
- Central Heating
- See all amenities
Cloonliss Lodge is a luxurious 4 Bedroomed Holiday Home ideally situated in the Woodlands of Mount Talbot, Co. Roscommon - where you will find relaxing nature walks. 4 Star Approved by Failte Ireland (Irelands Tourist Board) - 2 King Bedrooms with ensuites, 1 Double Bedroom and a Twin Bedroom. Sleeps 8 comfortably. …
- Pet friendly
- Smoking allowed
- Suitable for children
- Suitable for the elderly
- Wheelchair accessible
7 guest reviews
100% of guests would stay again
Superb house in a great setting
Left on Oct 23, 2013 for a stay in October 2013
I can only but commend Margaret for such a well kept and beautiful rental property. It was immaculately turned out and every room has such enchanting features! I've stayed here once previously and I had forgotten just how comfortable and warm the house is. I stayed this time with 6 x other for a family wedding in the locality. This house gave us a great base to travel to and from the event … See more
MacC D, Dublin, Ireland
Amenities included
- Television
- Parking
- Telephone
- Wireless Internet
- Towels Provided
- Soap/Shampoo Provided
- Books
- Radio
- Garden
- Computer Available
- See all amenities
Run by Rita Morgan, Armcashel is a comfortable, bright, spacious, non-smoking B&B; overlooking Clonalis Estate--the Ancestral Home of the O'Connor Clan. Situated just 1 km from Castlerea town centre on the Castlebar/Westport road. (N60) An elevated dormer house with green fields all around. A homely atmosphere and the…
- Pet friendly
- Smoking allowed
- Suitable for children
- Suitable for the elderly
- Wheelchair accessible
1 guest review
100% of guests would stay again
Best B & B I have ever stayed in!
Left on Jul 10, 2011 for a stay in June 2011
We were going to be a late check-in and Rita was very accommodating for our arrival. She had hot water bottles under the covers to warm the bed each night. My brother and I wanted to play golf and she arranged golf clubs and a course nearby to play. We had scones and tea every evening, or something stronger if desired. The property was within walking distance of town and very clean. It was … See more
norris_mh, Washington, Georgia
BALLYGLASS THATCHED COTTAGE, pet friendly, character holiday cottage, with a garden in Roscommon, County Roscommon, Ref 10139
- Bedrooms:
- 3
- Baths:
- 1
- Sleeps:
- 6
from$50/ night
Amenities included
- Washing Machine
- Television
- Towels Provided
- Refrigerator
- Linens Provided
Sleeping six people in three bedrooms, this quirky detached thatched cottage is set in an isolated location near Ballyglass, County Roscommon. Ballyglass Thatched Cottage is a charming detached property, full of character, in the countryside of County Roscommon, 8 miles from Roscommon town. The cottage has a ground floor…
- Pet friendly
- Smoking allowed
- Suitable for children
- Suitable for the elderly
- Wheelchair accessible
1 guest review
100% of guests would stay again
Old Charm
Left on Jun 29, 2013 for a stay in June 2013
Just returned home today, 29th June, after a weeks holiday. Quaint 150yr old cottage with deep walls, lovingly restored by the Fallon family. Set in farmland on traditional Irish narrow roads. We had also taken our chocolate labrador with us. Whilst pets are not allowed in the ground floor bedroom, there was plenty of room in the lounge area for her kennel. Great raised grass area for her to run… See more
paul s, newtownards
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Chandler Slough opens to public
Monday, October 6th, 2008Hikers, hunters, campers, anglers and horseback riders have a new place to enjoy along the Kissimmee River.
The South Florida Water Management District opened the 3,700-acre Chandler Slough on Sept. 20.
Slough land is covered with live oaks, pines, palms, bay and holly trees. There's a thick wetland shrub. Deer, wild turkey, wild hogs, gray foxes, alligators and several species of wading birds frequent the land.


text: Publications Search Results Your query returned 1 citations. Results 1 - 1 Results per page: 25 50 100 Sort by: Pub Date (newest) Title Recent Additions Author [1] Clear All Pub. Year Citation 1986 Goldsmith, Harold F., Jackson, David J., Kramer, Morton, Brenner, Berthold, Stiles, D.J., Tweed, D.L., Holzer, C.E., MacKenzie, E. Strategies for investigating effects of residential context: A study of depressed mood and major depression. Research on Aging. 8, (4), 609-635. Full Text Options: DOI Worldcat Google Scholar Export Options: RIS/EndNote Results 1 - 1 of 1 [1]


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Ideas for Teaching
Vignettes are stand-alone, illustrated electronic case studies that teach about geomorphology, surface processes, and/or Quaternary history. They are short, place-based examples that allow instructors to customize their class' approach to learning. Click here to browse the full collection of Vignettes.
A Case Study: Geomorphic Effects of the 2009 Big Pole Fire, Skull Valley, UT
On August 6, 2009, a lightning strike caused the Big Pole Fire to break out in Skull Valley on private and Federal lands managed by the Bureau of Land Management, the US Forest Service, and the State of Utah. The ~43,923 acre fire affected the western Stansbury Mountains, located about 40 miles west of Salt Lake City, in Utah. This fire event provides a valuable opportunity to observe changes in the local semi-arid ecosystem and landscape.The Sandstone Pavement Pine Barren of Northeastern New York: A legacy of Fire and Ice
The physical environment of the sandstone pavements strongly influences vegetation distribution and ecosystem processes; such as surface water runoff, organic matter decomposition and nutrient cycling; and the effects of ecological disturbances such as wildfires and ice storms.Wildfires, Floods, and Sediment Delivery at the Wilderness-Urban Interface in Coastal Southern California
Wildfires pose recurrent, news-making threats to life, property and livelihood throughout the arid American West. They often also contribute substantially to hillslope erosion and flooding by increasing runoff and destabilizing slopes against natural erosive processes such as dry ravel, rainsplash, sheetwash, gullying and shallow landsliding.


text: No. 90B. Although I may look like just a big blockhead, I assure you that I am quite a gentle and loving boy! My name's Bruno, and I'm looking for a new family. I was found as a stray in October. Now I'm just chillin' till my new ohana is ready for me. I already know some simple commands, walk well on a leash and will do almost anything to earn a treat and a smile. Hopefully someone will appreciate me for my good looks, friendly personality and eager-to-please disposition. If you need a larger than life new best bud, please come on down and ask for Bruno!


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Duke Energy will be working this weekend at the intersection of Fifth and Walnut Streets to relocate underground electric facilities. This work is being performed in conjunction with the Cincinnati Streetcar project.
West Fifth Street will be closed between Vine Street and Main Street. Walnut Street will be closed between E. Sixth Street and E. Fourth Street.
This closure will be put in place on July 12, 2013 at 6 p.m. and will remain in place until July 15, 2013 at 6 a.m.
Walnut Street access to the Fountain Square Garage will be maintained, as will emergency traffic.
A marked detour will direct motorists around the closure site. Metro bus service will be detoured around the area.
Duke's work will be performed generally between 7 a.m. and 9 p.m., with minimally disruptive work taking place during nighttime hours.
Copyright 2013 WXIX. All rights reserved.
725 S. Floyd Street Louisville, KY 40203 502-585-2201 FCC Public File firstname.lastname@example.com 502-561-4140 EEO Report Closed Captioning


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Posted By siteadmin | Feb 22,2013
Meek chops it up with Sway on what comes with living a rapper lifestyle. -
Posted By siteadmin | Feb 19,2013
Meek Mill has just announced his 'Dreams Come True' North American spring tour! Check out all the dates & RSVP on FACEBOOK here: http://cncrt.ly/dreamscometruetour. Tickets go on sale Friday, February 22nd! -
Posted By siteadmin | Feb 19,2013
The kid Lil Snupe is a beast!
Featured Merch
I ain't even eat no breakfast or lunch this #bikelife like crack! Lol
01/20/2014 - 19:45
01/20/2014 - 19:44
That was history! #305 #mlkweekend
01/20/2014 - 19:38
Where da pack at? http://t.co/4esQqnqL0L
01/20/2014 - 18:47
Who got a banshee 4 me I'm Tryna hit that Mlk parade..... I need a nice banshee! http://t.co/Wjmaot44SS
01/20/2014 - 17:06


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A Los Angeles bomb squad officer rescued a man from his burning vehicle after a fiery freeway crash.
The comments below are member-generated and do not necessarily reflect the opinions of PoliceOne or its staff.
Be the first to comment on this article. |
Tenn. Trooper Lowell Russell hopes the surgery will enable him to get back on the job
Nassive Palmer, 30, became combative after an officer saw the man had outstanding warrants and tried arresting him
The survey has been conducted five times since 1973
A Los Angeles police officer was killed and another seriously injured when their squad car collided with a big rig


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By Jennifer Martin - email
JACKSON, MS (WLBT) - Miller Dent was a 19-year-old agriculture student at Mississippi State when he was drafted to serve in World War II. He joined the Marines and after training, he was sent off to the South Pacific.
"It was crowded. You were put in a section aboard the ship. You were given a bunk and sometimes I believe the bunks went up four bunks high in the hole I was in. It wasn't anything to do except sit and look at the ocean," said Dent.
Dent would be a replacement soldier with the 3rd Marine Division. One of his closest friends and fellow Marines, future All American and NFL quarterback, Charlie Conerly.
"We were together all through the war. He chose to go into Mortars," added Dent.
Both men were part of the weapons company. Dent specialized in 81 MM Mortars. In June 1944 they sailed for Kwajalin, part of the Marshall Islands.
"We were there to be Reserve for the 2nd Marines going in to Saipan," said Dent.
"We were put on LST's, and they had the amphibious tractors in the hole that we would go in because of the coral that surrounded the island. We were on the LST's 52 days. On the LST's you didn't have any quarters. You had to live topside," Dent said.
"Conerly and I put our cots together and made us a hootch you might say. That kind of wore on you," Dent explained.
Once the situation in Saipan was secure, they were sent to Guam.
"We were in the assault waves. That night it was like a big thunderstorm. You could see flashes. You didn't get much sleep that night," Dent said.
He barely made it to the shore.
"On the way in, ours got hung up on an obstacle. We were drawing artillery fire the driver was able to get us off the obstacle, but as we circled the next round hit where the obstacle was," Dent added.
They landed on the Western Beaches and it took them 20 days to reach the Northern Coast of the island.
"It was like an amphitheater with a high cliff around it. Lost a lot of men going up in those cliffs," Dent said.
Dent's division would stay even after victory on the island.
"They left our division on Guam and we set up there. Guam, Saipan, and Tinian, they made airbases for the B29's to bomb Japan; a little island called Iwo Jima was giving them problems," Dent explained.
It would be where the 3rd Marines would be headed next.
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Join Date: Apr 2005
Location: Lexington, Kentucky
Posts: 16,954
Teen wins mayor's race by two votes
Teen wins mayor's race by two votes
DETROIT (Reuters) - The teen who grabbed the national spotlight this week by becoming mayor-elect of Hillsdale, Michigan, after a write-in campaign didn't win by much. Michael Sessions, an 18-year-old student at Hillsdale High School, won by a mere two votes over 51-year-old incumbent Douglas Ingles, instead of the 64-vote victory initially reported. Hillsdale County officials declared the final tally of 670 to 668 on Thursday. Sessions waged a write-in campaign for election because he was too young to get his name on the official ballot before the filing deadline. The unofficial vote count, announced Tuesday night, was skewed by the fact that voting machines counted every write-in vote as one for Sessions, County Clerk Thomas Mohr told reporters. That meant ballots with only a stray mark, or those on which the candidate's name was left blank, were erroneously tallied in Sessions' favor, Mohr said. Ingles could not be reached for comment on whether he plans to seek a recount. Since Tuesday's election, Sessions has been in the media spotlight, with appearances on "Late Night with David Letterman" and several of the TV networks' morning shows. In an interview with CNN from New York on Friday, as he shrugged off his tiny margin of victory, Sessions said he had wanted the top job in Hillsdale, a town of about 9,000 people, since early last year. "We've got to revitalize the economy," he said of his hometown, where 160-year-old Hillsdale College is located. He also acknowledged that it will take some juggling, between schoolwork and politics, to successfully complete his four-year term. "I'll be a student from 7:50 to 2:30, and then I'll work on mayor stuff from 3:00 to 6:00," Sessions said. "My only interest is probably the city of Hillsdale," he added. "I've never been a big partier." |
Join Date: Apr 2001
Location: Cincinnati, OH
Posts: 3,926
Re: Teen wins mayor's race by two votes
This thread is political in nature, if you wish to discuss it please do so on the other forum.
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Sundiros For Sale, Orange
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2007 Sundiro MopedScooter
6 days ago in Rancho Palos Verdes, CA
50mph, CA reg as Moped, You DO NOT NEED Yearly Registration, or Insurance, 50 miles, Like New, used ...