T7 RNA Polymerase Functions In Vitro without Clustering

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a Kd<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein

Confocal single-molecule fluorescence as a tool for investigating biomolecular dynamics in vitro and in vivo

Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo.Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis.To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.</p

Synthetic biology approaches to bio-based chemical production

Inexpensive petroleum is the cornerstone of the modern global economy despite its huge environmental costs and its nature as a non-renewable resource. While ninety percent of petroleum is ultimately used as fuel and can in principle be replaced by sources of renewable electricity, ten percent is used as a feedstock to produce societally important chemicals that cannot currently be made at a reasonable cost through alternative processes. In this dissertation, I will discuss my efforts, together with several colleagues, to apply synthetic biology approaches to the challenge of producing renewable petrochemical replacements. In Chapter 2, I discuss our efforts to engineer E. coli to produce fatty acids with a wide range of chain lengths at high yield, thereby providing an alternative platform for the production of diverse petrochemicals. In Chapter 3, I describe a novel method of DNA assembly that we developed to facilitate synthetic biology efforts such as those in Chapter 2. This method is capable of simultaneously assembling multiple DNA pieces with substantial sequence homology, a common challenge in synthetic biology. In Chapter 4, I discuss the development of a "bionic leaf": a hybrid microbial-inorganic catalyst that marries the advantages of photovoltaic-based light capture and microbial carbon fixation to achieve solar biomass yields greater than those observed in terrestrial plants. This technology offers a potentially low-cost alternative to photosynthesis as a source of biomass and derived chemicals and fuels. The work described in this dissertation demonstrates the capacity of synthetic biology to address the problem of renewable chemical production, and offers proof of principle demonstrations that both the scope and efficiency of biological chemical production may be improved

Confocal single-molecule fluorescence as a tool for investigating biomolecular dynamics in vitro and in vivo

Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Elongation complexes do not interact <i>in vitro</i> with a <i>K_d</i><1 µM.

<p><b>A.</b> The diffusion times (<i>τ_D</i>) of different fluorescently-labeled molecules (all at 2 nM; fluorophores indicated by purple circles) in buffer LS1, as determined by FCS using a single-species model. (i) Rhodamine 6G alone. (ii) A 70-bp template containing a T7 promoter, a 23-bp C-less cassette, and a C-containing 3′ end labeled with Cy3B. (iii) T7 RNAP ECs. A reaction containing labeled (2 nM) and unlabeled (0.1 µM) 70-bp templates was initiated by the addition of ATP+UTP+GTP, and incubated for 30 s to allow RNAPs to initiate on the templates and halt at the first C residues; then, the average diffusion time of the labeled templates was measured. (iv) As in (iii), except the unlabeled 70-bp template is replaced by an unlabeled 452-bp template encoding a T7 promoter, a C-less cassette, and a C-containing 3′ end (at 0.1–0.54 µM). This replacement does not significantly change the diffusion time of the labeled ECs, suggesting that they do not interact with unlabeled ECs. (v) Estimated diffusion time of the 452-bp template alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040207#pone.0040207.s005" target="_blank">Text S1C</a>). For all <i>τ_D</i> values, error was calculated using standard deviation (n≥3). <b>B.</b> Expected RNAP clustering. (i) An autocorrelation curve measured in the experiment of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040207#pone-0040207-g002" target="_blank">Fig. 2Aiv</a> (template and RNAP concentrations were 0.54 µM and 1.75 µM). Error bars represent standard deviation (n = 3). (ii) A fit of (i) using a single species model (Eq. 1); <i>τ_D</i> = 4.2 ms. (iii) The calculated autocorrelation function one would observe in the experiment (i), if RNAPs interacted with a <i>K_d</i> of 1 µM (calculated using a two-species model, Eq. 2). Sixty percent of labeled ECs diffuse freely with <i>τ_D</i> = 4 ms, while 40% are in RNAP dimers containing a 452-bp template, and so have a <i>τ_D</i> of 15 ms. This curve yields a <i>τ_D</i> of 6.0 ms when fit using a single-species model, and is clearly distinguishable from the measured data of (i). (iv) The autocorrelation function one would expect to observe in the experiment (i), were all labeled ECs to interact with a 452-bp template; all complexes have a <i>τ_D</i> of 15 ms.</p

Elongation complexes do not co-purify <i>in vitro</i>.

<p><b>A.</b> Strategy behind the ‘pulldown’ assay. T7 RNAP is mixed with three pieces of DNA (sample <i>1</i>): (i) an 800-bp promoter-less control fragment, (ii) a 452-bp template tagged with a 5′ biotin and encoding a <i>Bam</i>HI site, a T7 promoter, a C-less cassette, and a C-containing 3′ end, and (iii) a 290-bp template encoding a T7 promoter, C-less cassette, and C-containing 3′ end. After adding streptavidin beads, reactions were supplemented with ATP+UTP+GTP, and incubated for 30 s (to allow polymerases to initiate on the two templates and halt at the end of the C-less cassettes). If the now-engaged and halted polymerases interact, the 290-bp and 452-bp templates should associate (as shown). Next, beads (plus associated 290-bp and 452-bp templates) are pelleted, and the supernatant removed (sample <i>2</i>). Both supernatant and pellet are now treated with RNase and heated to 65°C to strip RNAPs and their transcripts from the templates (sample <i>3</i>); the pellet is also treated with <i>Bam</i>HI to release attached 452-bp templates from beads prior to analysis (sample <i>4</i>). If (elongating and halted) RNAPs interact (as shown), the 290-bp template (but not the 800-bp control fragment) should pellet with beads and the attached 452-bp template; then, the 290-bp template should be found in sample <i>4</i>. If they do not interact, the 290-bp template should not be found in the pellet (not shown). <b>B.</b> The assay described above was conducted in (i) 10 mM KCl (i.e., buffer LS1), (ii) 10 mM KCl plus tRNA (with 10-fold more tRNA than total template), and (iii) 100 mM potassium glutamate (i.e., buffer KGB); then, samples <i>1–4</i> were prepared, applied to ‘native’ 1.5% agarose gels, and the gels stained with SYBR green I. The 800-bp control fragment is present in samples <i>1–3</i>, but not <i>4</i> (as it fails to pellet). The 452-bp template is present in samples <i>1</i> and <i>4</i> (as it binds to beads, and pellets). Only trace amounts of the 290-bp template migrate as free DNA in sample <i>2</i> (elongation complexes migrate more slowly as a smear), but this amount is increased in sample <i>3</i> (as RNase and heat treatments release it from elongation complexes). The 290-bp template is found in sample 4 when the assay is performed in 10 mM KCl. However it is absent when the assay is performed in 10 mM KCl plus tRNA, or the more physiological buffer containing 100 mM K glutamate.</p

Validating the ‘3C strain’.

<p><b>A.</b> Diagram of the ‘3C strain’ genome. The strain expresses T7 RNAP (under control of a P_BAD promoter) in the presence of arabinose, but not in the presence of glucose. T7 RNAP then transcribes two transgenes driven by T7 promoters – <i>T7gene10</i> and <i>YFP</i> – integrated 100 kb apart in the bacterial chromosome. If active T7 RNAP clusters, these transgenes should interact in the presence of arabinose (i.e., when transcribed) but not glucose (i.e., when inactive). <b>B.</b> Validating ‘3C strain’. (i) Cells were grown to OD₆₀₀ = 0.4 in media containing arabinose or glucose, and their protein content analyzed using SDS-PAGE and western blotting. Probing for T7 RNAP shows it is expressed in the presence of arabinose (induction) but not glucose (repression). Probing for T7gp10 shows the same pattern, confirming that the corresponding gene is transcribed by T7 RNAP. In both blots, NusA is used as a loading control. (ii) Cells from the ‘3C strain’ imaged using fluorescence microscopy. YFP is detected when cells are grown in the presence of arabinose (+T7RNAP) but not glucose (−T7RNAP), confirming that its gene is transcribed by T7 RNAP. Both YFP images have the same intensity scale. Bar: 20 µm.</p

Genes transcribed by T7 RNAP do not interact <i>in vivo</i>.

<p><b>A.</b> Diagram of <i>Bgl</i>II fragments (thick coloured regions), PCR primers (yellow arrows) and ligation products (grey arrows) used in 3C. Ovals denote transcription units under the control of the T7 promoter. We determined the ligation frequency of the <i>Bgl</i>II fragment encoding <i>P_T7-T7gene10</i> (light blue; 24 kb) with (i) the 3-kb fragment encoding <i>P_T7-YFP</i> located 80 kb away (orange; using primers <i>a</i> and <i>c</i>), and (ii) a 5-kb promoter-less control fragment 22 kb away (purple; using primers <i>a</i> and <i>b</i>). As a secondary control for 3C efficiency, we also measured the ligation frequency of two adjacent fragments located at a randomly-chosen genomic site (pink and green; using primers <i>d</i> and <i>e</i>). This genomic map is drawn to scale. <b>B.</b> 3C. The ‘3C strain’ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040207#pone-0040207-g003" target="_blank">Fig. 3A</a> was grown to OD₆₀₀ = 0.4 in arabinose (+T7 RNAP) or glucose (−T7 RNAP), 3C templates prepared using <i>Bgl</i>II, and ligation products detected by PCR using the primer pairs indicated. Images show PCR products resolved on an agarose gel, and stained with SYBR green I. Loading controls (lanes 5–6) show that band intensities are proportional to the amount of ligation product present in the preceding PCR reactions, and thus to the contact frequency of the selected restriction fragments. All PCR products are of the expected size, and depend upon formaldehyde crosslinking (lane 3) and restriction nuclease digestion (lane 4). For each condition, the ‘test gene contact frequency’ (of <i>T7gene10</i> with <i>YFP</i>) was calculated by dividing the intensity of the band produced by the primers <i>a:c</i> by the intensity of the band produced by primers <i>a:b</i>. This adjustment corrected for differences in 3C efficiency, and so allowed ligation frequencies obtained from different samples (i.e., ± T7 RNAP) to be directly compared. Transcription of <i>T7gene10</i> and <i>YFP</i> by T7 RNAP had no effect on their contact frequency (compare test gene contact frequencies in lanes 1 and 2). Normalizing for 3C efficiency using the ligation frequency of the adjacent fragments (i.e., <i>a:c</i>/<i>d:e</i>) yielded the same result (1.9±0.6 a.u. for +T7 vs. 2.7±0.2 a.u. for −T7). a.u. = arbitrary units. *all measurements had standard deviations of less than 1% (n = 3). ^†assessed by qPCR.</p