Exploring conformational variability of an rna domain in the ribosome: from structure and function to potential antibiotic targeting

RNA in nature is modified at many specific sites to order to gain extra functions or to expand the genetic code. One of such RNAs is ribosomal RNA (rRNA), which contains several modified bases, particularly around the functionally significant sites. We have focused on understanding the influences of modified base on RNA structure and function by employing helix 69 (H69), which is a good region to evaluate the roles of modified bases since it contains three pseudouridines in the loop region and exists at the core of the ribosome. Previous model studies using small hairpin H69 showed the conformational differences of H69 loop under different conditions and revealed the significance of modified bases in H69 dynamics. Comparison of crystal structures of ribosomes indicates variable H69 conformations under different conditions. Based on these information, we performed dimethylsulfate (DMS) probing on 50S ribosomal subunits under different pHs, temperatures and Mg2+ concentrations, showing that H69 has a dynamic RNA loop component on the ribosome level as well as the models, and its multiple conformations are dependent on the presence of modified bases. In addition, footprintings in the presence of aminoglycoside antibiotics neomycin and paromomycin also show conformational variability in H69. These results indicate that H69 exists in multiple conformational states, which could be related to the function of the ribosome in the cell

Interactions between the Translation Machinery and a Translational preQ1 Riboswitch.

Gene expression is highly regulated with a diversity of regulation at the RNA level. In bacteria, regulation of mRNA translation into protein often occurs through RNA sequence features such as the Shine-Dalgarno (SD) sequence and local structural features. Translational riboswitches in bacteria exemplify such cis-acting regulation. This work look at how structural features of a preQ1 riboswitch effect regulation through interactions with the translation machinery. Broader questions about how individual translational machinery components, such as ribosomal protein S1 and the 30S ribosomal subunit, interact with structured RNAs are also addressed. We sought a more detailed mechanistic view of the interplay between the translational preQ1 riboswitch found in the 5′ UTR of an mRNA from T. tengcongensis, its ligand preQ1, and the SD sequence accessibility. To this end, we developed SiM-KARTS, a generalized strategy to interrogate site-specific structural dynamics of RNA molecules based on probe hybridization kinetics. Intriguingly, we found that the riboswitch expression platform alternates between conformations with differing SD accessibility, which are distinguished by “bursts” of probe binding, the pattern of which is modulated by ligand. This challenges the assumption that riboswitches behave in simple ON/OFF fashion and thus has broader implications for how we think about translational riboswitch regulation. The folding and unfolding of RNA structure influences other cellular processes besides translation. Ribosomal protein S1 performs other roles outside of the context of translation, which are related to its RNA binding or unfolding capacity. We used the well-characterized preQ1 riboswitch as a model pseudoknot to study how S1 interacts with defined, stable tertiary structure. S1 is able to bind and at least partially unfold this pseudoknot in a manner that is limited by RNA structural stability. Lastly, we investigated the influence of S1 on translation of preQ1 riboswitch-containing mRNAs and found that the effects of ligand on translation are not potentiated by the loss of S1. There is, however, a dramatic effect on translational coupling, invoking a role for S1 in polycistronic mRNA translation. These results highlight the need for additional techniques, such as assays at the single molecule level, to monitor early 30S-mRNA interactions during translation.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116677/1/palund_1.pd

Cell-Free Protein Synthesis

The Nobel Prize in Medicine 1968 for interpretation of the genetic code and its function in protein synthesis and in Chemistry 2009 for studies of the structure and function of the ribosome highlighted the ground-breaking experiment performed on May 15, 1961 by Nirenberg and Matthaei and their principal breakthrough on the creation of "cell-free protein synthesis (CFPS) system". Since then the continuous technical advances have revitalized CFPS system as a simple and powerful technology platform for industrial and high-throughput protein production. CFPS yields exceed grams protein per liter reaction volume and offer several advantages including the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists

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

rpsA and ribosomal protein S1: investigating a non-canonical translation initiation element

Translation initiation rates are fine-tuned by altering interactions between the ribosome and the translation initiation region of an mRNA. In bacteria varying levels of RNA structure attenuate these interactions by masking the ribosome-binding site from the small ribosomal subunit. Recent studies have described diverse strategies for recruiting mRNA to the ribosome, and highlighted the contributions of ribosomal protein S1. Here, I provide evidence that the non-canonical initiation mechanism that governs translation of the rpsA mRNA, encoding ribosomal protein S1, contains a three-dimensional architecture that is required for efficient translation. Furthermore, S1 plays an essential role during the initiation phase of translation by recruiting mRNA to the ribosome—unfolding structured mRNAs, and allowing for correct start codon positioning on the ribosome. Combining crosslinking immunoprecipitation and high-throughput sequencing approaches revealed the extent of S1’s involvement in mRNA recruitment, while also highlighting a broader role as a regulator of many RNA classes.Alberta Innovates (Strategic Chairs Program) [SC60-T2

Exploring Potential Drug Target Sites In The Ribosome Using Cisplatin And Its Analogues

Cis-diamminodichloridoplatinum (II), cisplatin, is an antitumor drug that has been used to treat several types of cancers. The reaction of cisplatin with DNA has been studied and discussed extensively in the literature; however, the effects of cisplatin on RNA function are poorly understood. In this thesis, two aspects of cisplatin, its preferred sites of interaction with RNA and its use as a chemical probe to gain accessibility information, were explored. To understand the site-selectivity of cisplatin with RNA, model RNA constructs and full-length 16S rRNA were employed. The binding studies revealed a cisplatin preference for guanosine-rich sequences. Primer extensions in 16S rRNA and MALDI-TOF in model constructs were used to locate the binding sites of cisplatin. HPLC and LC-MS were useful to determine the types and ratios of various adducts formed. Cisplatin and its analogues were employed to probe the accessibility of nucleotides on 16S rRNA, 30S subunits and 70S ribosomes in vitro as well as in vivo. This study revealed that many functionally important sites, such as helix 18, 24, 27, and 34 are accessible to the aquated platinum complex. Thus, these accessible sites can potentially be utilized as a new target sites in the design of structure-based antibiotics. When charge and size of the complex were changed, the binding preference was altered. In addition to the expected consecutive Gs, cisplatin analogues preferentially targeted AG sites on loop or bulge regions. Thus, several new complexes could be synthesized and utilized to gain more information about drug accessibility on the ribosome. The last part of the research focused on the application of siRNA to target non-Hodgkin\u27s lymphoma (NHL). Small interfering RNAs were designed to downregulate the c-Myc expression in NHL cells. Stabilities of designed siRNAs in media and their incorporation into liposomes were studied. Complexes of siRNA, liposomes, and antibody fragments (scFv) could be utilized in future applications to target specifically the c-Myc expression in NHL cells. Overall, this thesis work explored cisplatin binding to RNA and a number of possible new antibiotic target sites on the ribosome were identified. In the long term, further studies with fully functional ribosomes and comparisons with other organisms will have a greater impact on identifying novel drug target sites in pathogenic bacteria

Tuning ribosomal elongation cycle by mutagenesis of 23S rRNA

http://www.ester.ee/record=b1053388~S58*es

The Role of Initiation Factor Dynamics in Translation Initiation

Like most biological polymerization reactions, ribosome-catalyzed protein synthesis, or translation, can be divided into initiation, elongation, and termination stages. Initiation is the rate-limiting stage of translation and a critical site for translational control of gene expression. Throughout all stages of protein synthesis, the ribosome is aided by essential protein co-factors known as translation factors. I have studied the role that two translation initiation factors, IF1 and IF3, play in the mechanism and regulation of translation initiation in Escherichia coli. Specifically, I have used single-molecule fluorescence resonance energy transfer (smFRET) as a primary tool for investigating how the dynamics of IF1 and IF3 regulate the accuracy with which the translational machinery selects an initiator transfer RNA (tRNA) and the correct messenger RNA (mRNA) start codon during the initiation stage of protein synthesis