Plant luteoviral RNA viruses employ -1 frameshifting for the production of P1 and P1-P2 fusion proteins important for viral replication. Luteoviral pseudoknots are characterized by three adenosines in the 3' side of loop L2 known to be important for maintaining frameshifting efficiency and pseudoknot stability. A proposed P1-P2 mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) was of interest since it contained two adenosine to cytidine substitutions in L2. Functional analysis shows that the in vitro frameshifting efficiency is greater (~15%) than any other luteoviral pseudoknot. The NMR-derived solution structure of the ScYLV RNA pseudoknot shows that C25 is looped out of the triplex structure and the 3' most L2 cytidine (C27) and A24 form cis Watson-Crick/sugar-edge interactions with C14 and C15 in stem S1, respectively. Thus, the ScYLV pseudoknot maintains a similar triple helical architecture as other luteoviral pseudoknots. Surprisingly, the frameshifting efficiency of the C27A ScYLV pseudoknot is decreased by ~8 fold relative to wild-type ScYLV. The solution structure of the C27A ScYLV RNA exhibits a global fold similar to the wild-type RNA; however, distinct hydrogen bonding interactions at the helical junction are observed. Specifically, C8+ in the C8+ major groove base triple moves ~2.3 relative to the accepting (G12-C28) base pair relative to the WT RNA. New NMR experiments have been developed and/or applied to confirm Watson-Crick base pairs and tertiary structural interactions in the PEMV-1 and ScYLV pseudoknots by direct observation of trans hydrogen bond scalar couplings. In addition, intrabase couplings in cytidine and adenosine have been measured, providing a valuable tool for the assignment of amino and N3/N1 resonances in RNA. Finally, thermodynamic analysis of the pairwise coupling between the major groove and minor groove tertiary structural hydrogen bonds at the helical junction have been investigated by monitoring the thermal unfolding of WT, dC14, C27A, and dC14/C27A RNAs as a function of pH. Favorable pairwise coupling characterized the WT ScYLV and BWYV RNAs, while unfavorable coupling characterized the poorly functional C27A ScYLV RNA. The implications of these structural, functional, and thermodynamic findings on the mechanism of frameshift stimulation is discussed.

In this thesis I extend a class of grammars called conjunctive grammars to a stochastic form called stochastic conjunctive grammars. This extension allows the grammars to predict pseudoknotted RNA secondary structure. Since observing sec- ondary structure is hard and expensive to do with today's technology, there is a need for computational solutions to this problem. A conjunctive grammar can handle pseudoknotted structure because of the way one sequence is generated by combining multiple parse trees. I create several grammars that are designed to predict pseudoknotted RNA sec- ondary structure. One grammar is designed to predict all types of pseudoknots and the others are made to only predict a pseudoknot called H-type. These grammars are trained and tested and the results are collected. I am able to obtain a sensitivity of over 75% and a speci city of over 89% on H-type pseudoknots

In this thesis I extend a class of grammars called conjunctive grammars to a stochastic form called stochastic conjunctive grammars. This extension allows the grammars to predict pseudoknotted RNA secondary structure. Since observing sec- ondary structure is hard and expensive to do with today's technology, there is a need for computational solutions to this problem. A conjunctive grammar can handle pseudoknotted structure because of the way one sequence is generated by combining multiple parse trees. I create several grammars that are designed to predict pseudoknotted RNA sec- ondary structure. One grammar is designed to predict all types of pseudoknots and the others are made to only predict a pseudoknot called H-type. These grammars are trained and tested and the results are collected. I am able to obtain a sensitivity of over 75% and a speci city of over 89% on H-type pseudoknots

Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text

Mechanical unfolding of -1 frameshift signals such as RNA pseudoknots have aimed to test the hypothesis that the stability of the pseudoknot is directly correlated to the frameshifting efficiency. Here we report unfolding of the Beet Western Yellow Virus (BWYV) pseudoknot by optical tweezers experiments complemented by computer simulations using steered molecular dynamics (SMD). Seven pseudoknot scenarios were studied: the wild-type pseudoknot in the presence and absence of Mg<super>2+</super>, the wild-type pseudoknot at high pH (deprotonated C8), and C8U, C8A, A24G, G19U, and G19UC mutant constructs. The mutants were selected to probe three key structural features of the BWYV pseudoknot, a triple-stranded helix at the base of stem 1, the stem junction region of stem 1 and stem 2, and a unique quadruple base-pair interaction involving a protonated cytosine in position 8 (C8). These regions are thought to control ribosomal frameshifting by different strategies such as thermodynamic stability, kinetic influences, and dynamics involving contacts with the ribosome. In addition, the mutants have been shown to either abolish frameshifting ability of the pseudoknot (C8 mutant cases and A24G), or actually increase the frameshifting efficiency (as seen with G19U and G19UC). We find three major conclusions from the stretching of the pseudoknot constructs with optical tweezers. First, stretching in the absence of Mg<super>2+</super> results in no observed unfolding transitions. We interpret this to mean that magnesium is indispensible for the stable folding of the pseudoknot. Second, we found that frameshifting efficiency is not correlated with the force required to unfold the pseudoknots. However, we observe the unfolding of stem 1 in all of the pseudoknots stretched, where stem 2 unfolding is below our noise level. For this reason, we cannot rule out the possibility that an estimate of the thermodynamic stability of the entire pseudoknot would correlate with frameshifting efficiency. And third, we found that each pseudoknot mutant that resulted in reduced frameshifting efficiency also exhibited more off-equilibrium unfolding transitions that the wild-type pseudoknot under comparable loading rates. We conclude from these studies that the resistance of a pseudoknot to unfolding is controlled by both thermodynamic and kinetic parameters. We then suggest new technologies that would allow for greater resolution in order to correlate pseudoknot unfolding behavior with -1 programmed ribosomal frameshifting events.

No description available.

The ribosome is one of the most universal molecular machinery, synthesizing proteins in all living systems. The small ribosomal subunit plays a crucial role in decoding the messenger RNA during translation. We propose and validate a new architectural model of the ribosomal small subunit, with broad implications for function, biogenesis and evolution. We define an rRNA domain: compact and modular, stabilized by self-consistent molecular interactions, with ability to fold autonomously when it is isolated from surrounding RNA or protein. Each rRNA helix must be allocated uniquely to a single domain. These criteria identify a core domain of small subunit rRNA (domain A), which acts as a hub, linking to all other domains by A-form helical spokes. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2’OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with autonomous folding, and therefore classification as a domain. We show that the domain concept is applicable and useful for understanding the small ribosomal subunit. Our results support the utility of the concept of the domain as applied to at least some RNAs, the interdependence of the elements of domain A, and its ability to fold autonomously. Moreover, domain A, which exhibits elements of tRNA mimicry, is the essential core of the small ribosomal subunit. Understanding the structure and dynamics of domain A will provide valuable insight into the translational machinery.

The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.

Motivation: Improving the prediction of structures, especially those containing pseudoknots (structures with crossing base pairs) is an ongoing challenge. Current alignment-based prediction algorithms only find the consensus structure, and their alignments can come from structure-based alignment algorithms, which is more reliable, but come with an increased cost compared to sequence-based alignment algorithms. This step can be removed; however, non-alignment based algorithms neglect structural information that can be found within similar sequences. Results: We present a new method for prediction of RNA pseudoknotted secondary structures that combines the strengths of MFE prediction and alignment-based methods. KnotAli takes an RNA sequence alignment and uses covariation and thermodynamic energy minimization to predict secondary structures for each individual sequence in the alignment. We compared KnotAli's performance to that of three other alignment-based algorithms, on a large data set of 10 families with pseudoknotted and pseudoknot-free reference structures. We produced sequence alignments for each family using two well-known sequence aligners (MUSCLE and MAFFT). We found KnotAli to be superior in 6 of the 10 families for MUSCLE and 7 of the 10 for MAFFT. We find KnotAli's predictions to be less dependent on alignment quality. In particular, KnotAli is shown to have more accurate predictions compared to other leading methods as alignment quality deteriorates. Availability: The algorithm can be found online on Github at https://github.com/mateog4712/KnotAli / Graduate / 2022-08-16

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