Peter V. Cornish

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B Reveals Separation of Innate Immune Signaling Pathways

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC −, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC−. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.

Fluorescence enhancement from nano-gap embedded plasmonic gratings by a novel fabrication technique with HD-DVD

We demonstrate strong electromagnetic field enhancement from nano-gaps embedded in silver gratings for visible wavelengths. These structures fabricated using a store-bought HD-DVD worth

Structured mRNA induces the ribosome into a hyper-rotated state

10 and conventional micro-contact printing techniques have shown maximum fluorescence enhancement factors of up to 118 times when compared to a glass substrate under epi-fluorescent conditions. The novel fabrication procedure provides for the development of a cost-effective and facile plasmonic substrate for low-level chemical and biological detection. Electromagnetic field simulations were also performed that reveal the strong field confinement in the nano-gap region embedded in the silver grating, which is attributed to the combined effect of localized as well as propagating surface plasmons.

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

During protein synthesis, mRNA and tRNA are moved through the ribosome by the process of translocation. The small diameter of the mRNA entrance tunnel only permits unstructured mRNA to pass through. However, there are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here, we show using single molecule Förster resonance energy transfer and small angle X‐ray scattering experiments a new global conformational state of the ribosome. In the presence of the frameshift inducing dnaX hairpin, the ribosomal subunits are driven into a hyper‐rotated state and the L1 stalk is predominantly in an open conformation. This previously unobserved conformational state provides structural insight into the helicase activity of the ribosome and may have important implications for understanding the mechanism of reading frame maintenance.

Single-molecule Detection in Nanogap-embedded Plasmonic Gratings

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation

We introduce nanogap-embedded silver plasmonic gratings for single-molecule (SM) visualization using an epifluorescence microscope. This silver plasmonic platform was fabricated by a cost-effective nano-imprint lithography technique, using an HD DVD template. DNA/ RNA duplex molecules tagged with Cy3/Cy5 fluorophores were immobilized on SiO2-capped silver gratings. Light was coupled to the gratings at particular wavelengths and incident angles to form surface plasmons. The SM fluorescence intensity of the fluorophores at the nanogaps showed approximately a 100-fold mean enhancement with respect to the fluorophores observed on quartz slides using an epifluorescence microscope. This high level of enhancement was due to the concentration of surface plasmons at the nanogaps. When nanogaps imaged with epifluorescence mode were compared to quartz imaged using total internal reflection fluorescence (TIRF) microscopy, more than a 30-fold mean enhancement was obtained. Due to the SM fluorescence enhancement of plasmonic gratings and the correspondingly high emission intensity, the required laser power can be reduced, resulting in a prolonged detection time prior to photobleaching. This simple platform was able to perform SM studies with a low-cost epifluorescence apparatus, instead of the more expensive TIRF or confocal microscopes, which would enable SM analysis to take place in most scientific laboratories.

Studies on the Mechanisms of Translocation and Termination

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.

Operative Binding of Class I Release Factors and YaeJ Stabilizes the Ribosome in the Nonrotated State

This chapter addresses two long-standing questions concerning ribosome structure and function: (i) How are the mRNA and tRNAs moved through the ribosome following formation of each peptide bond? and (ii) how does recognition of a stop codon result in hydrolysis of peptidyl-tRNA? Not surprisingly, results from structural biology have played an important part in formulating mechanistic models for both of these processes. Although structural information is essential for understanding the detailed molecular mechanisms of such processes, it is in itself insufficient for establishing whether or not they are correct. There are already sufficient published examples of false mechanistic inferences based on ribosome structures to remind us that such models need to be tested experimentally, preferably by diverse approaches. Key aspects of the standard models for translocation and termination have emerged from structural observations — cryoEM reconstructions and x-ray crystallography, respectively. Both models have been subjected to experimental tests of various kinds, a process that continues in many laboratories. In the first part of this chapter, we describe the results of experiments using both single-molecule and bulk fluorescence methods to examine the relationship between intersubunit movement, hybrid-states binding of tRNA a6nd translocation. In the second part, we discuss a model for the mechanism of translation termination based on the x-ray crystal structures of the translation termination complexes, and some experimental tests of the model.

Single Molecule Studies of RNA-RNA Interactions

During translation, the small subunit of the ribosome rotates with respect to the large subunit primarily between two states as mRNA is being translated into a protein. At the termination of bacterial translation, class I release factors (RFs) bind to a stop codon in the A-site and catalyze the release of the peptide chain from the ribosome. Periodically, mRNA is truncated prematurely, and the translating ribosome stalls at the end of the mRNA forming a nonstop complex requiring one of several ribosome rescue factors to intervene. One factor, YaeJ, is structurally homologous with the catalytic region of RFs but differs by binding to the ribosome directly through its C-terminal tail. Structures of the ribosome show that the ribosome adopts the nonrotated state conformation when these factors are bound. However, these studies do not elucidate the influence of binding to cognate or noncognate codons on the dynamics of intersubunit rotation. Here, we investigate the effects of wild-type and mutant forms of RF1, RF2, and YaeJ binding on ribosome intersubunit rotation using single-molecule Förster resonance energy transfer. We show that both RF1 binding and RF2 binding are sufficient to shift the population of posthydrolysis ribosome complexes from primarily the rotated to the nonrotated state only when a cognate stop codon is present in the A-site. Similarly, YaeJ binding stabilizes nonstop ribosomal complexes in the nonrotated state. Along with previous studies, these results are consistent with the idea that directed conformational changes and binding of subsequent factors to the ribosome are requisite for efficient termination and ribosome recycling.

Ribosome Structure and Dynamics by smFRET Microscopy

Non-coding RNAs including microRNAs, siRNAs, and snoRNAs interact with their targets directly through RNA-RNA interactions by base-paring (van Himbergen et al., Nucleic Acids Res 21(8):1713-1717, 1993). RNA-RNA interactions play important roles in gene transcription and protein translation, which can be investigated with several experimental techniques including single molecule methods. Here, we describe how single molecule Förster resonance energy transfer (FRET) can be used to study RNA-RNA interactions in vitro by either surface immobilization or vesicle encapsulation.