A Computational Investigation on the Connection between Dynamics Properties of Ribosomal Proteins and Ribosome Assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins

Probing the Assembly of the Ribosome: Insights from Computational Studies on Ribosomal Proteins

Ribosomes are complex cellular machines that synthesize new proteins in the cell. The accurate and efficient assembly of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) to form a functional ribosome is important for cell growth, metabolic reactions, and other cellular processes. Ribosomal assembly has been an active research topic for many years because understanding the assembly mechansims can provide insight into protein/RNA recognitions that are important in many other cellular processes, as well as help optimize the development of antibacterial therapeutics. Experimental and computational sutdies thus far have greatly improved our understanding of assembly, yet many questions remain unanswered regarding the complex behaviors of r-proteins and rRNA during the process. To further understand ribosome assembly, we have computationally studied the sequences, structures, and dynamic properties of r-proteins from the 30S subunit and their relationships to RNA binding. We discuss the statistically greater amount of positively charged residues in r-proteins compared to other housekeeping proteins and observe a high level of charged interactions between r-proteins and rRNA in the assembled structure. We also detect a significant correlation between the overall flexibility of a protein and the number of contact points it makes with its rRNA binding site. Protein residues contacting with rRNA are observed to be more mobile in solution when compared to the non-contacting residues. We also describe common modes of structural dynamics, revealing likely conformational changes the proteins make prior to binding, how they relate to possible binding mechanisms used during the assembly and to the location of the protein in the fully assembled ribosome

Protein folding on the ribosome studied using NMR spectroscopy

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome-nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity

E. coli metabolic protein aldehydealcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosom

An RNA story, from chromatin to protein

RNA functions expand well over just coding for protein effectors. Non-coding RNAs oversee translation, RNA processing, and spatial organization of the cellular space, therefore participating in the regulation of almost any cellular process. To add a further layer of complexity, both coding and non-coding RNAs are extensively modified. These modifications determine RNA stability and localization, often by changing the way the single RNA molecules interact with RNA-binding proteins. In paper I, we describe how RNA contributes to maintaining an open chromatin structure by neutralizing the positive charge on the histone-tails. This effect, which seems to depend only on the charge of the RNA molecule, is not mediated by newly transcribed RNAs, but rather on RNA species that are stable in time, and possibly coincide with LINE- 1 containing transcripts. In paper II, we explore the role of dyskerin, the only RNA-guided pseudouridine synthase expressed by human cells, in mediating co-transcriptional modification of mRNAs, which in turn regulates their translation. Dyskerin travels along RNA polymerase II over transcribed genes, and binds – possibly modifying – thousands of mRNAs. After a shortterm depletion of dyskerin, pseudouridylation levels on mRNAs drop dramatically, and translation levels show an overall increase. We show that this effect depends on the enzymatic function of dyskerin and that pseudouridine directly inhibits translation in vitro. Conversely, we find that prolonged removal of dyskerin results in an overall drop in translational rates, and that this is linked to rRNA processing defects caused by long-term depletion of dyskerin. Our results also reveal that mRNA pseudouridylation is reduced in cells from dyskeratosis congenita patients, where dyskerin is impaired, therefore offer novel insight on the molecular mechanism behind this syndrome. In paper III, we investigate the role of mRNA pseudouridylation in the heat-shock response, focusing on the formation of stress granules. Pseudouridine levels increase after heat-shock, and removal of three different enzymes involved in mRNA pseudouridylation results in defective stress granule formation. Hypo-pseudouridylated mRNAs accumulate within stress granules, and translation recovery after stress is impaired. Taken together, these results expand on the multi-faceted role of RNA and RNA modification in regulating multiple fundamental cellular processes. The insight they offer on the inner workings of human cells and the molecular mechanism behind dyskeratosis congenita will hopefully contribute to the identification of novel therapeutic targets for dyskeratosis congenita and other diseases

Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments

RNAs are crucial for many cellular functions. Thus, studying ligand-RNA interactions and their dynamics in response to changes in the surrounding environment is important. In spite of the well-known DNA coordination, current research also indicates cisplatin binding to RNA. Kinetic studies of rRNA platination reactions are largely unexplored. This research was conducted to achieve two objectives. First, a broad kinetic study was carried out to investigate the cisplatin-rRNA interactions. The structure, function, and ligand interactions depend on RNA microenvironments. Second, the application of platination kinetics as a tool to interrogate RNA electrostatic environments was explored. Three model rRNA hairpins from E. coli ribosome were selected. Two helix 69 (H69) constructs, modified H69 (with pseudouridine) and unmodified H69 (without pseudouridine), and the 790 loop, which has an identical size and nucleotide composition to unmodified H69, were used. Prior to kinetic studies, cisplatin targets on each RNA were determined using RNase T1 mapping combined with MALDI MS, and dimethyl sulfate (DMS) probing. The kinetic studies were carried out under pseudo-first-order conditions and electrostatic properties were evaluated using Brønsted-Debye-Hückel and polyelectrolyte theories. RNase T1 mapping with MALDI MS and dimethyl sulfate (DMS) probing revealed GpG sites as cisplatin targets on RNA. The DMS probing further revealed platination-induced structural changes in RNA. Both the RNA sequence and modified nucleotides showed an impact on platination rates. Kinetic data showed that the platination rate is dependent on cations and the abundance of active cisplatin complexes. Structure, pseudouridylation, availability of active cisplatin species, and cation/Pt+ electrostatic competitions all impact platination of the two H69 RNAs. Probing neomycin-H69 interactions by platination kinetics indicated that structural changes in modified H69 upon aminoglycoside binding could also impact the platination kinetics. Electrostatic models revealed that nucleotide sequence, cations, and H+ ions impact the global RNA electrostatics. The similar global electrostatic properties between the two H69 RNAs indicated that structure-dependent electrostatic changes in modified H69 could be limited to the loop region. In conclusion, this thesis work showed that both intrinsic RNA characteristics such as structure, sequence, and dynamics, as well as bulk solution conditions (e.g. cations and pH), impact cisplatin-RNA interactions. The RNA electrostatic parameters determined in this thesis work illustrated platination kinetics can be used as an informatory tool for probing dynamic RNA microenvironments