Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR

Solid-state NMR of non-crystalline recombinant viral particles

High-resolution solid-state Nuclear Magnetic Resonance (ssNMR) is rapidly emerging as a powerful structural tool in chemistry and biology and is applicable to a wide range of problems that cannot be addressed by solution state NMR or X-ray crystallography. Steady ongoing methodological developments combined with tremendous advances in probe and spectrometer hardware have led to a variety of strategies for resonance assignment, paving the way to the first 3D structure determinations of a wide range of samples at atomic resolution, ranging from inorganic frameworks and catalysts to membrane proteins and fibrils. Solid-state NMR does not suffer from molecular weight limitations (unlike its solution counterpart) and can be applied to non-crystalline samples. Therefore, this method is uniquely positioned to answer key questions about the chemistry and the structure of macromolecular assemblies which, because of their size and structural flexibility, are often difficult to characterize. However several important problems remain to be solved before ssNMR is ready to cope with challenging solid biochemical assemblies, and many methodological developments are still expected in this fast evolving field. The principal goal of this thesis has been to test a panel of experimental ssNMR methodologies for the structural investigation of large non-crystalline RNA-protein complexes such as determining the architecture and function of viral capsids. In detail, we have focused on three highly relevant biological systems, the nucleocapsids of Measles virus (MeV), of Acinetobacter phage 205 (AP205), and of Rice Yellow Mottle Virus (RYMV). These molecules constitute a very challenging target whose molecular weight is 10 to 100 times larger than any structural study previously performed on globular proteins by ssNMR. In these viral particles, genomic RNA is protected by multiple copies of a coat protein (the so-called N-protein, of about 400 amino acids for MeV), which are organized into complex superstructures (helical for MeV, and icosahedral for AP205 and RYMV, respectively). These systems are too large for solution state NMR, and the difficulty to obtain large single crystals prevents conventional X-rays analysis. However, recombinant (13C,15N)-labelled nucleocapsids can be prepared, and low resolution structures obtained by electron microscopy are available. In the present work, solid-state NMR was used to study the nucleoproteins in the capsid superstructure. The repetitive positioning of the coat proteins provide precisely the degree of order necessary for high resolution NMR spectra. We have optimized sample conditions such that the proteins can be studied in situ, and have recorded the first (13C,13C), (15N,13C) and (1H, 31P)-correlation spectra (notably, with the aid of high magnetic fields and ultra-fast magic angle sample spinning), which constitute the first steps towards resonance assignment and structure determination, including determination of the quaternary interaction between neighboring proteins on the RNA. The analysis of the NMR data was enriched by a series of circular dichroism (CD) spectra, which probe the relative arrangements of aromatic bases in the RNA, and of peptide groups along the capsid protein backbone. The work is highly interdisciplinary, going from research in virology and molecular biology to quantum chemistry, via structural biology and bioinformatics

Twister ribozyme : from synthetic biology to in vivo function

The recent description of a new widespread class of small endonucleolytic ribozymes termed “twister” on one hand provides new opportunities for the development of advanced tools for RNA engineering and on the other hand opens essential questions on the biological utility of this and other classes of small-self cleaving motifs presenting similar distributions and genetic contexts. The present doctoral dissertation explores the potential of the twister ribozyme as a biotechnological tool for the construction of novel ribozyme-based synthetic switches and investigates its role in messenger RNA (mRNA) processing and posttranscriptional regulation in bacteria. The first part of the thesis proposes twister ribozyme as an outstandingly flexible expression platform, which in conjugation with two different aptamer domains enables the construction of different regulators of gene expression in bacteria. First, a series of one-input dependent riboswitches are generated that control gene expression at the translational level, via the sequestration of the ribosome binding site. The sensor domains (theophylline and thiamin pyrophosphate aptamers) are attached to two different sites of the catalytic core of the ribozyme using in vivo screened communication modules. Then, in vivo Boolean logic gates that act at the translational level are developed taking advantage of the fact that the twister ribozyme scaffold offers at least two independent sites for attaching ligand-sensing aptamer domains. The in vivo screening stands out as an ideal strategy to generate compact two-input riboswitches that sense and simultaneously respond to two small molecular signals and that can be described as AND, NAND, OR, NOR, ANDNOT and ORNOT binary Boolean operators. Interestingly, the described design mimics the situation observed in some naturally occurring twister ribozymes where multiple optional domains are directly connected to the catalytic core in positions P1, P3 and P5. Two-input riboswitches are also generated using a novel design in which a TPP aptamer is connected to the theophylline aptamer in an “in-series” fashion. In the second part of the dissertation, several bacterial twister motifs, previously identified in intergenic regions and presenting different configurations and structural organizations, are characterized using both in vitro and in vivo assays. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. Our data show that the ribozyme-mediated cleavage of the bacterial 3’- untranslated region (3’-UTR) has major effects on gene expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they show to depend primarily on the motif-specific structural features and on mRNA stabilization properties of the secondary structures that remain on the 3’-UTR after ribozyme cleavage. Using these principles, novel artificial twister-based riboswitches are developed that exert their switching activity via ligand-dependent cleavage of the 3’-UTR and the removal of the protective intrinsic terminator stem-loop. The fact that these artificially developed switches can effectively regulate gene expression relying on the cleavage of the 3’-UTR suggests that similar ligand-dependent modulation of the ribozyme activity could exist also in nature with possible functions in gene expression regulation. Overall, this dissertation describes the experimental procedures and design approach to employ the twister ribozyme for the construction of new tools that have the potential of being broadly applied in biotechnology, synthetic biology and metabolic engineering. The evidences deriving from the characterization of a number of bacterial twister ribozymes and the observed versatility in our artificial gene-regulatory setups are used to discuss possible natural roles of this newly discovered and widespread catalytically active motif in bacteria.publishe

Ligand-dependent ribozymes

The discovery of catalytic RNA (ribozymes) more than 30 years ago significantly widened the horizon of RNA-based functions in natural systems. Similarly to the activity of protein enzymes that are often modulated by the presence of an interaction partner, some examples of naturally occurring ribozymes are influenced by ligands that can either act as cofactors or allosteric modulators. Recent discoveries of new and widespread ribozyme motifs in many different genetic contexts point toward the existence of further ligand-dependent RNA catalysts. In addition to the presence of ligand-dependent ribozymes in nature, researchers have engineered ligand dependency into natural and artificial ribozymes. Because RNA functions can often be assembled in a truly modular way, many different systems have been obtained utilizing different ligand-sensing domains and ribozyme activities in diverse applications. We summarize the occurrence of ligand-dependent ribozymes in nature and the many examples realized by researchers that engineered ligand-dependent catalytic RNA motifs. We will also highlight methods for obtaining ligand dependency as well as discuss the many interesting applications of ligand-controlled catalytic RNAs.publishe

The 3′-untranslated region of mRNAs as a site for ribozyme cleavage-dependent processing and control in bacteria

Besides its primary informational role, the sequence of the mRNA (mRNA) including its 5′- and 3′- untranslated regions (UTRs), contains important features that are relevant for post-transcriptional and translational regulation of gene expression. In this work a number of bacterial twister motifs are characterized both in vitro and in vivo. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. Our data show that the ribozyme-mediated cleavage of the bacterial 3′-UTR has major effects on gene expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they show dependence on motif-specific structural features and on mRNA stabilization properties of the secondary structures that remain on the 3′-UTR after ribozyme cleavage. Using these principles, novel artificial twister-based riboswitches are developed that exert their activity via ligand-dependent cleavage of the 3′-UTR and the removal of the protective intrinsic terminator. Our results provide insights into possible biological functions of these recently discovered and widespread catalytic RNA motifs and offer new tools for applications in biotechnology, synthetic biology and metabolic engineering.publishe

A nascent polypeptide sequence modulates DnaA translation elongation in response to nutrient availability

The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes; nevertheless, the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA, we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, is critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that translation regulation by nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions

A predictive model for the ichnological suitability of the Jezero crater, Mars: searching for fossilized traces of life-substrate interactions in the 2020 Rover Mission Landing Site.

none5siopenBaucon A, Neto de Carvalho C, Briguglio A, Piazza M, Felletti FBaucon, A; Neto de Carvalho, C; Briguglio, A; Piazza, M; Felletti,

Phosphate starvation decouples cell differentiation from DNA replication control in the dimorphic bacterium Caulobacter crescentus.

Upon nutrient depletion, bacteria stop proliferating and undergo physiological and morphological changes to ensure their survival. Yet, how these processes are coordinated in response to distinct starvation conditions is poorly understood. Here we compare the cellular responses of Caulobacter crescentus to carbon (C), nitrogen (N) and phosphorus (P) starvation conditions. We find that DNA replication initiation and abundance of the replication initiator DnaA are, under all three starvation conditions, regulated by a common mechanism involving the inhibition of DnaA translation. By contrast, cell differentiation from a motile swarmer cell to a sessile stalked cell is regulated differently under the three starvation conditions. During C and N starvation, production of the signaling molecules (p)ppGpp is required to arrest cell development in the motile swarmer stage. By contrast, our data suggest that low (p)ppGpp levels under P starvation allow P-starved swarmer cells to differentiate into sessile stalked cells. Further, we show that limited DnaA availability, and consequently absence of DNA replication initiation, is the main reason that prevents P-starved stalked cells from completing the cell cycle. Together, our findings demonstrate that C. crescentus decouples cell differentiation from DNA replication initiation under certain starvation conditions, two otherwise intimately coupled processes. We hypothesize that arresting the developmental program either as motile swarmer cells or as sessile stalked cells improves the chances of survival of C. crescentus during the different starvation conditions

Twister ribozymes as highly versatile expression platforms for artificial riboswitches

The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class.publishe