Preferential location of bulged guanosine internal to a G.C tract by 1H NMR

A series of double-helical oligodeoxyribonucleotides of sequence corresponding to a frame-shift mutational hot spot in the lambda CI gene, 5'-dGATGGGGCAG, are compared by proton magnetic resonance spectroscopy at 500 MHz of the exchangeable protons. Duplexes containing an extra guanine in a run of two, three, and four G.C base pairs are compared to regular helices of the same sequence and to another sequence containing an isolated bulged G, 5'-dGATGGGCAG.dCTGCGCCATC. The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Resonances assigned to the G tract in bulge-containing duplexes are shifted anomalously upfield and are very broad. Imino proton lifetimes are determined by T1 inversion-recovery experiments. The exchange rates of G-tract imino protons in bulged duplexes are rapid compared to those in regular helices and are discussed in terms of the apparent rate of solvent exchange for the isolated G bulge. Delocalization of a bulged guanosine in homopolymeric sequences can explain the observed changes in chemical shift and relaxation times across the entire G.C run, and the chemical shifts can be fit by a simple model of fast exchange between base-paired and unpaired states for the imino protons. This allows us to calculate the relative occupancies of each bulge site. In these sequences, we find the extra base prefers positions internal to the G tract over those at the edge.     

RNA folding and ribosome assembly

Ribosome synthesis is a tightly regulated process that is crucial for cell survival. Chemical footprinting, mass spectrometry, and cryo-electron microscopy are revealing how these complex cellular machines are assembled. Rapid folding of the rRNA provides a platform for protein-induced assembly of the bacterial 30S ribosome. Multiple assembly pathways increase the flexibility of the assembly process, while accessory factors and modification enzymes chaperone the late stages of assembly and control the quality of the mature subunits.     

Role of counterion condensation in folding of the Tetrahymena ribozyme. II. Counterion-dependence of folding kinetics

Condensed counterions contribute to the stability of compact structures in RNA, largely by reducing electrostatic repulsion among phosphate groups. Varieties of cations induce a collapsed state in the Tetrahymena ribozyme that is readily transformed to the catalytically active structure in the presence of Mg2+. Native gel electrophoresis was used to compare the effects of the valence and size of the counterion on the kinetics of this transition. The rate of folding was found to decrease with the charge of the counterion. Transitions in monovalent ions occur 20- to 40-fold faster than transitions induced by multivalent metal ions. These results suggest that multivalent cations yield stable compact structures, which are slower to reorganize to the native conformation than those induced by monovalent ions. The folding kinetics are 12-fold faster in the presence of spermidine3+ than [Co(NH3)6]3+, consistent with less effective stabilization of long-range RNA interactions by polyamines. Under most conditions, the observed folding rate decreases with increasing counterion concentration. In saturating amounts of counterion, folding is accelerated by addition of urea. These observations indicate that reorganization of compact intermediates involves partial unfolding of the RNA. We find that folding of the ribozyme is most efficient in a mixture of monovalent salt and Mg2+. This is attributed to competition among counterions for binding to the RNA. The counterion dependence of the folding kinetics is discussed in terms of the ability of condensed ions to stabilize compact structures in RNA.     

The effect of long-range loop-loop interactions on folding of the Tetrahymena self-splicing RNA

Bas?e-pairing between the terminal loops of helices P2.1 and P9.1a (P13) and P2 and P5c (P14) stabilize the folded structure of the Tetrahymena group I intron. Using native gel electrophoresis to analyze the folding kinetics of a natural pre-RNA containing the Tetrahymena intron, we show that P13 and P14 are the only native loop-loop interactions among six possible combinations. Other base-pairing interactions of the loop sequences stabilize misfolded and inactive pre-RNAs. Mismatches in P13 or P14 raised the midpoints and decreased the cooperativity of the Mg(2+)-dependent eqXuilibrium folding transitions. Although some mutations in P13 resulted in slightly higher folding rates, others led to slower folding compared to the wild-type, suggesting that P13 promotes formation of P3 and P7. In contrast, mismatches in P14 increased the rate of folding, suggesting that base-pairing between P5c and P2 stabilizes intermediates in which the catalytic core is misfolded. Although the peripheral helices stabilize the native structure of the catalytic core, our results show that formation of long-range interactions, and competition between correct and incorrect loop-loop base-pairs, decrease the rate at which the active pre-RNA structure is assembled.     

Structural requirement for Mg2+ binding in the group I intron core

Divalent metal ions are required for splicing of group I introns, but their role in maintaining the structure of the active site is still under investigation. Ribonuclease and hydroxyl radical footprinting of a small group I intron from Azoarcus pre-tRNA(Ile) showed that tertiary interactions between helical domains are stable in a variety of cations. Only Mg(2+), however, induced a conformational change in the intron core that correlates with self-splicing activity. Three metal ion binding sites in the catalytic core were identified by Tb(III)-dependent cleavage. Two of these are near bound substrates in a three-dimensional model of the ribozyme. A third metal ion site is near an A minor motif in P3. In the pre-tRNA, Tb(3+) cleavage was redirected to the 5' and 3' splice sites, consistent with metal-dependent activation of splice site phosphodiesters. The results show that many counterions induce global folding, but organization of the group I active site is specifically linked to Mg(2+) binding at a few sites.     

Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA

Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.     

Chlorophyll f-driven photosynthesis in a cavernous cyanobacterium

Chlorophyll (Chl) f is the most recently discovered chlorophyll and has only been found in cyanobacteria from wet environments. Although its structure and biophysical properties are resolved, the importance of Chl f as an accessory pigment in photosynthesis remains unresolved. We found Chl f in a cyanobacterium enriched from a cavernous environment and report the first example of Chl f-supported oxygenic photosynthesis in cyanobacteria from such habitats. Pigment extraction, hyperspectral microscopy and transmission electron microscopy demonstrated the presence of Chl a and f in unicellular cyanobacteria found in enrichment cultures. Amplicon sequencing indicated that all oxygenic phototrophs were related to KC1, a Chl f-containing cyanobacterium previously isolated from an aquatic environment. Microsensor measurements on aggregates demonstrated oxygenic photosynthesis at 742 nm and less efficient photosynthesis under 768- and 777-nm light probably because of diminished overlap with the absorption spectrum of Chl f and other far-red absorbing pigments. Our findings suggest the importance of Chl f-containing cyanobacteria in terrestrial habitats.The textbook concept that oxygenic phototrophs primarily use radiation in the visible range (400–700 nm) has been challenged by several findings of unique cyanobacteria and chlorophylls (Chl) over the past two decades (Miyashita et al., 1996; Chen et al., 2010; Croce and van Amerongen, 2014) Unicellular cyanobacteria in the genus Acaryochloris primarily employ Chl d for oxygenic photosynthesis at 700–720 nm (Miyashita et al., 1996) and thrive in shaded habitats with low levels of visible light but replete of near-infrared radiation (NIR, >700 nm, Kühl et al., 2005; Behrendt et al., 2011, 2012). Furthermore, Chl f was recently discovered in filamentous (Chen et al., 2010; Airs et al., 2014; Gan et al., 2014) and unicellular cyanobacteria (Miyashita et al., 2014), enabling light harvesting even further into the NIR region up to ∼740 nm, often aided by employing additional far-red light-absorbing pigments such as Chl d and phycobiliproteins (Gan et al., 2014). Whereas the biochemical structure (Willows et al., 2013) and biophysical properties (Li et al., 2013; Tomo et al., 2014) of Chl f have been studied in detail, the actual importance of this new chlorophyll for photosynthesis is hardly explored (Li et al., 2014).Chlorophyll f has been found in cyanobacteria originating from aquatic/wet environments: the filamentous Halomicronema hongdechloris from stromatolites in Australia (Chen et al., 2012), a unicellar morphotype (Strain KC1) from Lake Biwa in Japan (Akutsu et al., 2011; Miyashita et al., 2014) and a filamentous Leptolyngbya sp. strain (JSC-1, Gan et al., 2014) from a hot-spring and in a unicellular Chlorogloeopsis fritschii strain from rice paddies (Airs et al., 2014). In this study, we report on a unicellular Chl f-containing cyanobacterium originating from a wet cavernous habitat and demonstrate its capability of NIR-driven oxygenic photosynthesis. Enrichments of the new cyanobacterium were obtained from a dense dark green-blackish biofilm dominated by globular morphotypes of Nostocaceae growing on moist limestone outside Jenolan Caves, NSW, Australia. The sampling site was heavily shaded even during mid-day with low irradiance levels of 400- to 700-nm light varying from 0.5 to 5 μmol photons m⁻² s⁻¹. Biofilms were carefully scraped off the substratum and kept in shaded zip-lock bags in a moist atmosphere until further processing. Samples were then incubated at 28 °C in a f/2 medium under NIR at 720 nm (∼10 μmol photons m⁻² s⁻¹) yielding conspicuous green cell aggregates after several months of incubation. Repeated transfer of the aggregates into fresh medium resulted in a culture predominated by green cell clusters (Figure 1a), exhibiting orange-red fluorescence upon excitation with blue light (Figure 1b). Transmission electron microscopy revealed that the green clusters consisted of slightly elongated unicellular cyanobacteria (∼1- to 2-μm wide and ∼2- to 3-μm long), with stacked thylakoids and embedded in a joint polymer matrix (Figure 1c). Hyperspectral microscopy (Kühl and Polerecky, 2008) of the clusters revealed distinct troughs in the transmission spectra at absorption maxima indicative of Chl a (675–680 nm) and Chl f (∼720 nm; Figure 1d, red line). In situ spectral irradiance measurements at the sampling site showed strong depletion of visible wavelengths in the 480- to 710-nm range (Figure 1d, gray line), whereas highest light levels were found in the near-infrared region of the solar spectrum at 710–900 nm. The presence of Chl a and f was further confirmed in enrichment cultures using high-performance liquid chromatography-based pigment analysis (Figure 1e, Supplementary Figure S1), while no Chl d was detected. In addition, weak spectral signatures of carotenoids and phycobilins, with absorption occurring at ∼495 and 665 nm, were evident in the hyperspectral data. Cyanobacteria, including those producing Chl d/f, are known to actively remodel their pigment content in response to the available light spectrum (Stomp et al., 2007; Chen and Scheer, 2013; Gan et al., 2014) and Chl d/f has almost exclusively been found in cyanobacteria grown under far-red light and not under visible light (Kühl et al., 2005; Chen et al., 2010; Airs et al., 2014; Gan et al., 2014; Li et al., 2014; Miyashita et al., 2014). Recent work describes this acclimation response as ‘Far-Red Light photoacclimation'' (FaRLiP), which, in strain JSC-1, comprises a global change in gene expression and structural remodeling of the PSII/PSI core proteins and phycobilisome constituents (Gan et al., 2014). The extent to which this arrangement results in optimized photosynthetic performance is only known for the NIR (=710 nm)-acclimated strain JSC-1, where exposure to wavelengths >695 nm resulted in 40% higher O₂ evolution rates as compared with cells that were previously adapted to red light (645 nm; Gan et al., 2014). Yet the discrimination of actinic wavelengths and their relative effect on gross photosynthesis in Chl f-containing cells needs further investigation. Using an O₂ microsensor and the light–dark shift method (Revsbech et al., 1983) on embedded Chl f-containing aggregates, we found maximal gross photosynthesis rates (∼1.06 μmol O₂ cm⁻³ s⁻¹) to occur at irradiances of ∼250 μmol photons m⁻² s⁻¹ of 742 nm (half-bandwidth, HBW, 25 nm, Figures 2a and b) with light saturation to occur very early at ∼35 μmol photons m⁻² s⁻¹. Further red-shifted actinic light, that is, 768 nm (HBW 28 nm) and 777 nm (HBW 30 nm), yielded lower O₂ evolution rates, which, in all likelihood, are an effect of the diminished overlap with far-red light-absorbing pigments, including Chl f (Figures 2a and b). As O₂ evolution rates were measured on non-axenic cell aggregates, 16S rDNA amplicon sequencing was employed to determine the microbial diversity found within the enrichment culture. This revealed the presence of a variety of bacterial types, including anoxygenic phototrophs, yet all sequences for known oxygenic phototrophs in the data set (∼9.3% of all reads on the order level, Supplementary Figure S2) formed a single operational taxonomic unit (OTU) closely affiliated with the Chl f-containing strain KC1 (Miyashita et al., 2014, Figure 2c).Open in a separate windowFigure 1Imaging and pigment analysis of Chl f-containing cyanobacteria isolated from a cavernous low-light environment. (a) Representative bright field microscope image of cultured cells grown under 720 nm NIR. (b) Fluorescence image of the same cells as in a, excited at 450–490 nm, with emission being detected at >510 nm. (c) Transmission electron microscopy of a Chl f-containing cyanobacterium with densely stacked thylakoid membranes. (d) Transmittance spectrum of cell aggregate determined by hyperspectral imaging (red line). Ambient light conditions at the site of isolation (gray line), as measured by a spectroradiometer. Note the Chl f-specific in vivo absorption at ∼720 nm in the transmittance spectrum (dotted line). Small insert picture denotes the cells and area of interest (black arrow) from which the spectrum was taken. (e) In vitro absorption spectrum of Chl f extracted from enrichment cultures and analyzed via high-performance liquid chromatography. The two Chl f-specific absorption peaks (404 and 704 nm in acetone:MeOH solvent) are indicated.Open in a separate windowFigure 2Taxonomic affiliation and O₂ evolution of Chl f-containing cells as determined by O₂ microelectrode measurements and 16 S rDNA amplicon sequencing. (a) Emission spectra of narrow-band light-emitting diodes (LEDs) used in this study, with peak emissions at 742, 768 and 777 nm indicated by a–c, respectively. (b) Gross photosynthesis measured via an O₂ microsensor placed in a clump of agarose-embedded Chl f-containing cells. Different NIR irradiance was administered by the LEDs in a and by altering the distance of the LEDs to the embedded cells. (c) Phylogenetic affiliation of known Chl f and/or Chl d-containing cyanobacteria (highlighted in gray) and their respective habitat/place of isolation. Taxonomy was determined by clustering all known oxygenic phototrophs found in enrichment cultures from this study (at order level) into a single OTU (=292 bp length, see Supplementary Materials for details). Phylogeny was inferred using Maximum-likelihood in conjunction with the GTR +I +G nucleotide substitution model, tree stability was tested using bootstrapping with 100 replicates. The analysis involved 39 nucleotide sequences each truncated to a length of 292 bp. Here, the green-sulphur bacterium Chlorobium tepidum TLS was chosen as the outgroup.This advocates that cells from our enrichment culture are related to KC1 cells and supports, in conjunction with further morphological-, physiological- and ultrastructural evidence, that Chl f is extending the usable light spectrum for oxygenic photosynthesis in a cavernous low-light environment. Given the lifestyle and known habitats of recognized Chl d/f-producing cyanobacteria (Figure 2c), we propose that many, if not all, surface-associated cyanobacteria are intrinsically capable of producing far-red light-absorbing pigments and to actively employ them in oxygenic photosynthesis as a result of FaRLiP or similar, yet unknown, mechanisms.