Torsional restraint: a new twist on frameshifting pseudoknots

mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot

Co-translational genetic switching during protein synthesis: the HIV-1 Nef gene as a paradigm

The old maxim of “one gene, one mRNA, one protein” no longer holds, especially with viral genes. It is possible for one mRNA to encode several proteins of unrelated functions in overlapping reading frames of a single oligonucleotide, or for an additional protein domain to be added on to a protein at the C-terminal by the readthrough of a stop codon. The question of how and when stop does not always mean stop, how slippage from one reading frame into another is controlled, and the factors that trigger those genetic switches, are the subjects of this research. The focus of the project is the HIV-1 nef gene, which has examples of both of these types of co-translational switching events (translational frameshifting and stop codon readthrough). Nef is a myristoylated protein expressed in the early stage of the HIV-1 life cycle, which functions as a fundamental factor for efficient viral replication and pathogenesis. One of the notable features of nef is the highly conserved 3’-UGA stop codon, and the potential for the protein to be extended by about 30 amino acids if readthrough of that stop codon can occur. We hypothesize that antisense tethering interactions (ATIs) between viral mRNA and host selenoprotein mRNA enables capture of the host selenocysteine insertion sequence (SECIS) element to enable the expression of virally encoded selenoprotein modules via translation of in frame UGA stop codons as selenocysteine (SeC). This mRNA hijack mechanism was predicted theoretically using computational analysis and was experimentally supported at the DNA level by gel shift assay. Readthrough of UGA was proved at the mRNA level by fluorescence microscopy image analysis and flow cytometry of transfected HEK 293 cells with engineered reporter gene plasmid vector constructs, in which the downstream reporter gene can only be expressed if the UGA is translated. siRNA knockdown of thioredoxin reductase 1 (TR1) mRNA in transfected cells resulted in decreased GFP expression, consistent with the hypothesis that host-virus mRNA tethering may enable selenocysteine incorporation for the stop codon readthrough. Furthermore quantitative analysis of TR1 mRNA knockdown demonstrated using RT-PCR confirmed that the siRNA treatment results in approximately 20% knockdown of TR1. The HIV-1 nef coding region features a potential -1 frameshift site with a potential overlapping gene region near the middle of the coding sequence. A sequence matching the pattern (XXXYYYZ) of a known -1 frameshifting “slippery sequence” signal is present in the nef sequence at this point, immediately upstream of a G-quadruplex (QPX) sequence that serves to regulate frameshifting. An in vitro frameshift assay using a dual reporter vector was constructed, in which the putative HIV-1 nef-fs sequence with QPX was cloned between two fluorescent reporter genes. Cells transfected with this construct showed orange fluorescence, which is only possible if the -1 frameshifting occurs. Treating the transfected cells with QPX stabilizing synthetic drug TMPYP4 increased the frameshifting efficiency by 27%, specifically confirming the role of the QPX as an enhancer of -1 frameshifting efficiency

Study of complex RNA function modulated by small molecules: the development of RNA directed small molecule library and probing the S-adenosyl methionine discrimination between on and off conformational states of the SAM-I riboswitch

RNA recently remained unexploited and is now drawing interest as a potential drug target. The methodology and available drug libraries for RNA targeting/screening are in rudimentary stages. The interactions made by ligands with RNA can be explored for RNA based drug development. The dissertation is composed of 4 chapters. The first chapter focuses on the structural features of RNA and the attempts made to target RNA previously. The second chapter focuses on the development of a small molecule library enriched with substructures derived from RNA binding ligands. For this study a fragment-based approach (fragment based approach is detailed in chapter 2) is used in order to accommodate the conformational flexibility of RNA. The library molecules are used for screening against suitable RNA targets using NMR. We identified at least 5 ligands out of which 2 are novel ligands binding to the ribosomal 16s rRNA. The third chapter is focused on the role of small molecules in inducing conformational changes in an RNA genetic regulatory element called the S-Adenosyl methionine (SAM) SAM-I riboswitch. The mechanistic features of the SAM-I riboswitch to understand the basis for specificity and discrimination and its gene regulation mechanism are reported. To address the conformational dynamics Bacillus subtilis and Thermoanearobacter tencongenesis SAM-I riboswitches in response to SAM binding several conformer mimics are designed, synthesized and characterized using NMR, equilibrium dialysis, and inline probing. The study shows that apart from the conserved residues of the binding pocket, residues downstream of the binding pocket are involved in detecting SAM and assist the binding of SAM to the riboswitch with weak affinity. Our data highlights the capacity of a so-called antiterminator helix from the expression platform to assist the formation of a partial P1 helix of the aptamer domain. A stable P1 is involved in recognition and tight binding of SAM. Our in vitro experiments suggest that the riboswitch could switch from an unbound conformation to tightly SAM bound structure through weakly binding intermediate structures in the presence of the small molecule SAM. The future directions are included in the fourth chapter along with the conclusions

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

Encoding folding paths of RNA switches

RNA co-transcriptional folding has long been suspected to play an active role in helping proper native folding of ribozymes and structured regulatory motifs in mRNA untranslated regions. Yet, the underlying mechanisms and coding requirements for efficient co-transcriptional folding remain unclear. Traditional approaches have intrinsic limitations to dissect RNA folding paths, as they rely on sequence mutations or circular permutations that typically perturb both RNA folding paths and equilibrium structures. Here, we show that exploiting sequence symmetries instead of mutations can circumvent this problem by essentially decoupling folding paths from equilibrium structures of designed RNA sequences. Using bistable RNA switches with symmetrical helices conserved under sequence reversal, we demonstrate experimentally that native and transiently formed helices can guide efficient co-transcriptional folding into either long-lived structure of these RNA switches. Their folding path is controlled by the order of helix nucleations and subsequent exchanges during transcription, and may also be redirected by transient antisense interactions. Hence, transient intra- and intermolecular base pair interactions can effectively regulate the folding of nascent RNA molecules into different native structures, provided limited coding requirements, as discussed from an information theory perspective. This constitutive coupling between RNA synthesis and RNA folding regulation may have enabled the early emergence of autonomous RNA-based regulation networks.Comment: 9 pages, 6 figure

Stability and Kinetics of DNA Pseudoknots: Formation of T∗A•T Base-Triplets and Their Targeting Reactions

Pseudoknots have been found to play important roles in the biology of RNA. These stem-loop motifs are considered to be very compact and the targeting of their loops with complementary strands is accompanied with lower favorable free energy terms. We used a combination of spectroscopic (UV, CD and fluorescence), calorimetric (DSC, PPC and ITC) and kinetic (SPR) techniques to investigate: 1) Local base-triplet formation in pseudoknots; 2) energetic contributions for the association of pseudoknots with their complementary strands; and 3) the kinetic rates as a function of targeting strand length. We investigated a set of DNA pseudoknots with sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where “Tn” is a thymine loop with n = 5, 7, 9, and 11. The favorable folding of each pseudoknot resulted in favorable enthalpy-entropy compensation, correlated to favorable base-pair stacking contributions and unfavorable uptakes of ions and water molecules. The increase in the length of the loop yielded higher TMs, 53°C to 59°C and folding enthalpies ranging from -60 to -110 kcal/mol, resulting in a significant stabilization, ΔG°(5) = -8.5 to -16.6 kcal/mol, which is consistent with the formation of 1-2 TAT/TAT base-triplet stacks. The PPC results yielded folding volume changes, ΔVs, ranging from 18 to 23 ml/mol, indicating the higher volume of the folded pseudoknots is due to the uptake of both water (ΔnW of -11 to -24 mol H2O/mol) and ions (Δnion of -2.5 to -4.1 mol Na+/mol). We use ITC and DSC to determine thermodynamic profiles for the reaction of pseudoknots with partially complementary strands. We obtained favorable reaction free energies terms. However, the targeting of compact pseudoknots containing local base-triplets is less favorable due to their larger folding free energy term. The SPR data indicated that the rate of association, kon, decreases while the rate of dissociation, koff, increases as the length of the targeting strand increases, which yielded increasing KD, app.. This indicates the affinity of the target strand to the pseudoknot decreases as the length of the target strand increases. A similar trend was obtained when dissociation constants, KD, DSC, were measured from DSC Hess cycles. However, the KD, DSC were much smaller. This apparent discrepancy between these techniques is that SPR is measuring both the initial association and initial dissociation rates of steady state equilibrium states, while DSC measures true equilibrium states of the entire molecules

Trans-translation is an appealing target for the development of new antimicrobial compounds

Because of the ever-increasing multidrug resistance in microorganisms, it is crucial that we find and develop new antibiotics, especially molecules with different targets and mechanisms of action than those of the antibiotics in use today. Translation is a fundamental process that uses a large portion of the cell’s energy, and the ribosome is already the target of more than half of the antibiotics in clinical use. However, this process is highly regulated, and its quality control machinery is actively studied as a possible target for new inhibitors. In bacteria, ribosomal stalling is a frequent event that jeopardizes bacterial wellness, and the most severe form occurs when ribosomes stall at the 30-end of mRNA molecules devoid of a stop codon. Trans-translation is the principal and most sophisticated quality control mechanism for solving this problem, which would otherwise result in inefficient or even toxic protein synthesis. It is based on the complex made by tmRNA and SmpB, and because trans-translation is absent in eukaryotes, but necessary for bacterial fitness or survival, it is an exciting and realistic target for new antibiotics. Here, we describe the current and future prospects for developing what we hope will be a novel generation of trans-translation inhibitors