Dissertations / Theses on the topic 'Conserved RNA Structures'

The family Picornaviridae includes many important human pathogens. RNA structures play important roles in picornavirus molecular biology and recent evidence suggests that these are more extensive than previously thought. In this project we identified a number of potential RNA structures in picornavirus genomes and started to analyse one of these structures. The work focussed on human parechoviruses (HPe V). The structure of the HPe V 5' untranslated region (UTR) was analysed by obtaining several new sequences and using an alignment of 60 sequences to identify covariant changes. This allowed the previously predicted structure to be confirmed and refined. Aligned sequences representing most picornavirus species were then analysed for suppression of synonymous codon variation (SSCV). Strong SSCV was seen in several cases and this was often related to the presence of RNA structures including the Cre and novel potential structures. Patterns of conserved dinucleotides were also used to identify regions of importance in the picornavirus genome. A new program, Dinucleotider (1.0) was developed and used, which allows a graphical output of conserved dinucleotides in aligned sequences. CG was found to be the most informative dinucleotide and could be used to identify regions of the picornavirus genome, which corresponded to the 5'UTR, 3'UTR and Cre, as well as further new structures. Genetic analysis of a predicted structure in the 3D-encoding region of HPe V s, was carried out by making two mutants, with 3 or 6 mutations in one of the structural domains. Both sets of mutations had little effect on virus growth in cultured cells, suggesting that the structure does not play a critical role in replication and other possible roles need to be identified. Overall, this project has allowed several RNA structures to be identified in picornaviruses. These are conserved between related viruses and presumably play important roles in the biology of picornaviruses. They need to be studied further in order to improve understanding of how picornaviruses infect cells, which is required to improve diagnosis and control of these pathogens.

State-of-the-art methods in RNA secondary structure prediction focus on predicting the final, functional structure. However, ample experimental and statistical evidence indicates that structure formation starts immediately during transcription and this co-transcriptional folding influences the resultant final RNA structure. Thus, identifying the transient structures that are formed co-transcriptionally may bring insight into understanding how co-transcriptional folding leads to the final conformation in vivo. As RNA secondary structures are currently best predicted by comparative approaches, we therefore investigated whether homologous RNA genes not only assume the same final structure, but also share structural features during the co-transcriptional folding in vivo. For this, we compiled a non-redundant data set of 32 transcripts deriving from six different RNA families which constitutes the most comprehensive data set with experimentally confirmed transient and alternative RNA structures so far. We present statistical evidence that homologous RNA genes from related organisms fold co-transcriptionally in a similar way. In particular, we show that some transient structures are highly conserved with levels similar to those of the final, functional structure. Moreover, we find that the predicted co-transcriptional folding pathways of homologous sequences encounter similar transient structure features, which often coincide with known transient features. We thus also predict candidates for these evolutionarily conserved transient features of co-transcriptional folding pathways in silico. We further expand 4 alignments from the aforementioned dataset by searching via covariance model and manual curation in order to share them with the RNA community. These alignments either update the existing Rfam datasets with annotation of transient structures, or introduce new RNA family: (1) Trp operon leader, where alternative structures are coordinated to regulate the operon transcription in response to tryptophan abundance (2) HDV ribozyme, where the self-cleavage activity is modulated via transient structures involving the extended 5’ flanking sequence (3) 5’ UTR of Levivirus maturation protein, where a transient structure temporarily postpones the formation of the final structure that inhibits the translation of maturation protein (4) SAM riboswitch, where the downstream gene expression is regulated by alternative structures upon binding of SAM.
Science, Faculty of
Graduate

The secondary structure of RNA molecules is often critical to their proper functioning, and so prediction of those structures has been a focus of bioinformatics research for many years. RNA folds as it is transcribed, and it has lately become apparent that the sequences of structures that an RNA adopts (its folding path) is vitally important for the RNA to fold into its proper structure. Analysis of the evolution of a group of related RNAs is useful for identifying conserved and therefore functionally important secondary structures. In theory, functional but transient secondary structures which play a role in the folding pathway should also be detectable in this way. Moreover, folding may be affected by a variety of factors in the cellular environment, which presents a challenge to the existing methods of RNA pathway prediction via simulation. Evolutionarily conserved helices are implicitly the ones formed in vivo, and so is a useful means of accounting for this problem. Here we present TRANSAT, a method of identifying evolutionarily conserved elements of RNA secondary structure, including transient structures, from an alignment of related RNAs. We evaluate TRANSAT’s performance on a wide variety of alignments, present some examples of its predictions, and show how its predictions may be useful for predicting folding pathways. We also present a method of generating simulated alignments, and use these alignments to examine TRANSAT’s performance in ways that are challenging with alignments of real sequences.

The KsgA enzymes comprise an ancient family of methyltransferases that are intimately involved in ribosome biogenesis. Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution, and therefore were present in the last common ancestor. The first member of the family to be described, KsgA from Escherichia coli, was initially shown to be the determining factor for resistance/sensitivity to the antibiotic kasugamycin and was subsequently found to dimethylate two adenosines in 16S rRNA during maturation of the 30S subunit. Since then, numerous other members of the family have been characterized in eubacteria, eukaryotes, archaea and in eukaryotic organelles. The eukaryotic ortholog, Dim1, is essential for proper processing of the pre-rRNA, in addition to and separate from its methyltransferase function. The KsgA/Dim1 family bears sequence and structural similarity to a larger group of S-adenosyl-L-methionine dependent methyltransferases, which includes both DNA and RNA methyltransferases. In this document we report that KsgA orthologs from archaea and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life. We have characterized KsgA structurally, and discuss aspects of KsgA's activity in light of the structural data. We also propose a model for KsgA binding to the 30S subunit, based on solution probing data. This model sheds light on KsgA's unusual regulation and on the dual function of the Dim1 enzymes.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 126-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

In this project a number of peptidyl transferase antibiotics were studied, specifically a group of aminohexose cytosine nucleoside antibiotics and their interaction with a selected number of highly conserved ribonucleic acid (RNA) motifs, designed to represent their possible binding site within the ribosome. This group of antibiotics shows a wide range of interesting properties, including antiviral and anti-tumour activity, and as they bind to a particularly conserved region in the ribosome, they are likely to be difficult for microorganisms to develop resistance to. It is hoped that once the mechanism of action of these antibiotics is better understood, that modifications to the antibiotics can be effectively made to create new or hybrid antibiotics with more selective antibacterial, or indeed antiviral or anti-tumour properties. The nuclear magnetic resonance (NMR) structure of the RNA binding, peptidyl tranferase inhibitor antibiotics amicetin, blasticidin S and gougerotin, in their native solution states, have been successfully determined. The structures all exhibit a stable conformation, stabilised by intramolecular hydrogen bonds. Amicetin was observed to be folded, distinctly different from the linear, extended conformation of amicetin previously determined by X-ray crystallography. The structure of blasticidin S was found to be very similar to its X-ray crystal structure. Gougerotin was shown to form a similar conformation to blasticidin S, save that the end chain of gougerotin was bent at right angles to the rest of the molecule, forming a structure similar to that of the major bound X-ray crystal structure of blasticidin S. All the solution structures showed a similar conformation in the analogous regions of their chemical structure, suggesting that hybrid antibiotics could be produced.Two highly conserved RNA motifs of Halobacterium halobium (H. h.) and Escherichia coli (E. coli) 23S ribosomal RNAs were chosen to investigate their interaction with amicetin. The NMR structure of the H. h. and E. coli. 29-mer RNA motifs have been determined; the motifs both form well folded A-form RNA conformations. The E. coli NMR structure differs from the X-ray crystal structure of the motif contained within the ribosome, as a highly conserved adenine residue, which resides in a bulge strongly implicated with amicetin binding, folds into the helix as opposed to being flipped out. Instead, an adjacent cytosine residue partially flips out; whereas in the crystal structure, it is folded within the helix. The NMR stuctures of the H. h. motif differs from the X-ray crystal structure of the motif, contained within the ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. To continue this study, a fully 13C and 15N isotopically labelled version of the H. h. RNA sample has been partially assigned, and an initial structure determination has been performed, using ultra high field 1 GHz spectroscopy.Addition of amicetin to both the H. h. and E. coli 29-mer RNA samples were accompanied by discrete changes to the spectra, suggesting weak interaction between the two components. These can be qualitatively interpreted to changes induced in the local conformation of the RNA motifs and the amicetin arising from the formation of a complex, between the amicetin and the bulge region of the particular motif.

The peptidyl transferase centre (PTC) of 23S ribosomal RNA is the target for a number of antibiotics which inhibit protein synthesis. The precise mode of binding of these antibiotics is largely unknown and hence is an active area of research in structural biology. The NMR solution structures of three PT antibiotics, bamicetin, sparsomycin and anisomycin have been successfully characterised using a range of two-dimensional NMR techniques and restrained molecular dynamics. The NMR structures of the these antibiotics provided valuable first hand insight into their conformations, since no X-ray crystal structures of the antibiotics in their free states have been determined so far. Bamicetin adopts a folded conformation possibly held by intramolecular hydrogen bonds and similar to the published NMR structure of amicetin. These antibiotics generate spontaneous single nucleotide mutants upon prolonged exposure and bamicetin and sparsomycin are universal PT inhibitors, interacting with all three evolutionary domains of 23S rRNAs. The amicetin antibiotic produces a spontaneous single mutation U2457C in the Halobacterium halobium (H.hal) 23S rRNA and the binding site is predicted to be very close to this nucleotide. The similarity in chemical structure with amicetin, suggests bamicetin to target the same binding site on the 23S rRNA. Both bamicetin and sparsomycin show exchange retarded amide proton resonances in the NMR spectrum, akin to other amicetin family antibiotics, indicating the retarded exchange to be a characteristic feature in the native solution state. The Bacillus subtilis (B.subtilis) 70S ribosomes have strong affinity for bamicetin and so a highly conserved 27mer RNA motif containing the possible binding site was selected for NMR structure determination and bamicetin binding studies. The greater number of imino proton resonances observed together with the high quality of the determined structure of the motif proved that B.subtilis rRNA is more stable than E.coli and H.hal rRNAs. The B.subtilis 27mer rRNA-bamicetin interaction studies revealed a fast exchange, weak binding system and careful analysis of line width and chemical shifts indicated changes at the local conformation of the RNA after binding. To probe the cross-hypersensitivity phenomenon, a 25mer RNA corresponding to the thiostrepton-resistant mutant (G1159) residing in the domain II of H.hal 23S rRNA was chosen for NMR structure determination and amicetin binding. Discrete chemical shift changes and NOESY experiments using ultrahigh field 1GHz NMR revealed weak interactions. The structures of the antibiotics and analysis of their dynamics as well as interactions with the RNA motifs of different organisms have yielded important information in understanding their binding and inhibitory activities at the atomic level. The results can be used for generating new or hybrid antibiotics to tackle the escalating problem of antibiotic resistance.

The conserved secondary structural RNA motifs of EncephaloMyoCarditis Virus (EMCV) have been well characterised biochemically and shown to play an important role in translation initiation by a novel cap-independent mechanism called Internal Ribosomal Entry Site (IRES). However, the three dimensional structure and interactions of these conserved motifs are not known, and hence the mechanism is not fully understood. The NMR results described in this thesis have provided, for the first time, new structural knowledge on the conformation of these motifs, their affinity for Mg2+ and their intermolecular interactions. RNA motifs selected from two separate domains (I and J) of the IRES structure were investigated using a range of 2D and 3D NMR techniques. The apical ‘hammerhead’ region of the I domain contains a highly conserved 16mer RNA which hosts a stable and mutationally sensitive G547CGA550 tetraloop. Sequence specific assignments were carried out on this motif, along with its Mg2+ complex, and a large number of NMR experimental constraints were generated for the RNA structure determination. Similarly, high resolution NMR structures of a distal 17mer RNA, which has been predicted to be a potential receptor for the GCGA tetraloop, and its Mg2+ complex were also produced. Thus, we were able to demonstrate that Mg2+ stabilises the RNA tertiary structure via non-specific interactions. Since the largest changes were induced at the tetraloop motif, we propose that Mg2+ stabilises the 16mer into an optimum conformation which is essential for IRES function. The determination of the structures of the above motifs led us to investigate the 16mer-17mer binary (1:1) complex at 1 GHz, in the presence of Mg2+. Significant changes were observed in the 1H and 31P chemical shift, NOE intensity and line width, clearly demonstrating RNA-RNA interactions taking place between the two components. The most interesting result to emerge was the distinct absence of NOEs from G547{NH} of the stable tetraloop, thus highlighting an important structural role for this functionally critical residue. Since no previous work has shown a clear interaction between the two RNAs, the results obtained in this project provide the first direct experimental evidence for intramolecular interactions in the I domain of EMCV IRES.Finally, we show how isotopically labelled RNAs can be successfully used as an aid in NMR assignment, analysis and structure determination. The J domain of EMCV IRES binds to eIF4GII protein and is essential for translation initiation. A suite of 3D NMR techniques were carried out on a highly enriched and uniformly 13C, 15N-labelled 39mer RNA. Several key features of the RNA, which may be involved in protein recognition, were identified. Further, a selectively 19F-labelled 16mer RNA from the I domain, was also studied to show how fluorine NMR can be used to probe RNA structure, dynamics and interactions. The RNA motifs of the EMCV IRES were shown to exhibit high stabilities, which are brought about by the complex folding of the various secondary structural elements involving RNA- Mg2+, RNA-RNA and RNA-protein tertiary interactions. It is these vital interactions that enable the IRES to recruit the ribosome in the translation initiation step of protein synthesis, and have laid a strong foundation for further NMR investigation of the whole IRES.

Picornaviruses are important causes of human illness and it is necessary to understand more about how these viruses function. Human parechoviruses (HPeV) are common pathogens and studies have shown that 95% of people become infected with HPeV at a very early age, usually with symptoms such as mild diarrhoea and fever. However, one virus type HPeV3, is implicated in much more serious cases of neonatal disease and so it is important to understand HPeVs to increase the opportunity to develop drugs or vaccines against the infection. The HPeV1 genome encodes a single polyprotein that is cleaved into structural and non-structural proteins. Analysis of one region of the genome (encoding the polymerase, 3Dpol) shows that some codons are perfectly conserved, suggesting functions in addition to protein coding. This region seems to fold into an RNA secondary structure made up of three stem-loops and a tertiary structure “kissing” interaction. The structure was validated by comparing all the available HPeV sequences and found to be highly conserved. To investigate if the structure has a role in RNA stability, an EGFP fluorescent assay was used. Sequences containing the structure was added to the 3’ UTR of the EGFP gene. A mutant with 21 mutations which completely destroys the RNA structure was also used. A FACS-based method was used to measure expression levels of EGFP. The results showed that there was a significant reduction in fluorescence from the mutant construct. The effect of the structure was also investigated in infected cells and in cells exposed to different stresses which could mimic virus infection. The results suggest that the structure can positively affect RNA stability/translation. Further investigation on other possible roles such as RNA replication and translation should be performed to improve the understanding of the biology of the structure in HPeVs and a Renilla Luciferase reporter gene system was assembled to facilitate the studies in the future.

The structure, kinetics, and interactions of the conserved 16mer and 15mer RNA motifs of the internal ribosome entry site (IRES) of the Foot-and-Mouth Disease virus (FMDV), have been investigated by homonuclear and heteronuclear NMR techniques. The 16mer RNA is endowed with a classic GNRA tetraloop motif, which is essential for IRES activity and the 15mer RNA motif is a potential tetraloop receptor. We have determined three high resolution NMR solution structures of the 16mer apo-RNA, the 16mer Mg2+ RNA complex and the 15mer apo-RNA with RMSDs of 0.17Å, 0.16Å and 0.35Å, respectively. The high precision of these NMR structures was achieved by including a large number of NMR experimental restraints, derived from NOEs and coupling constants, and validating them using the MolProbity program. The 16mer RNA structure comprised of six base pairs with a GUAA tetraloop and the 15mer RNA structure comprised of four base pairs and a large heptaloop; this is the first heptaloop to be studied by NMR.Addition of Mg2+ to the 16mer apo-RNA caused selective chemical shift changes to the stem G177 and loop G178 imino proton resonances, suggesting Mg2+-induced conformational change to the GUAA tetraloop. This was supported by a significant chemical shift change to the selectively 19F-labelled loop U179 in the 5-FU 16mer RNA. Furthermore, variable temperature experiments revealed retarded imino proton exchange for the stem and loop imino protons, demonstrating the enhanced thermodynamic stability conferred by Mg2+. This enhancement in stability was confirmed by measuring the imino proton exchange rates for the 16mer apo-RNA and the 16mer Mg2+ RNA complex. Analysis of the 16mer apo-RNA and its Mg2+ RNA complex NMR solution structures revealed that Mg2+-induced structural changes to the GUAA tetraloop act to stabilise the loop via stronger base stacking and intramolecular interactions. Fascinatingly, we discovered that Mg2+ ions provide increased stability required for the formation of a G.A sheared base pair in the GUAA tetraloop. RNA-RNA interactions between the 16mer and 15mer RNAs and their fluorinated analogues were studied by NMR spectroscopy. Small changes to chemical shift and linewidth of proton peaks in the non-fluorinated RNA-RNA complex provided evidence for a weak interaction between the loop of the 16mer RNA and the stem of the 15mer RNA. 19F-NMR experiments revealed additional peaks for the 19F-labelled U179 of the fluorinated 16mer/15mer RNA complex providing further good evidence of RNA-RNA interaction. The NMR structures of the conserved RNA motifs and their interactions have yielded important information in understanding the properties and behaviour of RNA. This will provide the first stepping stone in understanding the IRES mechanism and its use in antiviral therapy and biotechnology.

Orientador: Nilson Ivo Tonin Zanchin
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A síntese de ribossomos é um processo conservado em eucariotos e se inicia com a transcrição dos rRNAs no nucléolo. Mais de 170 fatores atuam de forma transitória no processamento dos precursores para gerar os rRNAs maduros que formarão as subunidades ribossomais no citoplasma. Entre as proteínas envolvidas na síntese de ribossomos está a Nip7p, uma proteína nucleolar de 21 kDa associada ao complexo pré-60S em Saccharomyces cerevisiae. Nip7p é conservada e possui ortólogas em eucariotos e em Archaea. A análise da seqüência primária revela a presença de um domínio conservado na região C-terminal, denominado PUA, encontrado em diversas proteínas associadas a modificações no RNA. Neste trabalho, foram realizadas análises estruturais e funcionais com o objetivo de investigar a função molecular da proteína Nip7 no processamento e modificação do rRNA. A estrutura tri-dimensional de PaNip7, ortóloga de Nip7p em Pyrococcus abyssi foi resolvida por difração de raios-X até 1,8Å de resolução, utilizando o método SIRAS. Comparação estrutural seguida por ensaios in vitro confirmaram o envolvimento do domínio PUA na interação com RNA. Além disso, tanto Nip7p como suas ortólogas PaNip7 e HsNip7 interagem com seqüências ricas em uridina, indicando que atuam de forma semelhante no processamento do rRNA. Essa preferência por uridina pode ainda explicar a afinidade da proteína Nip7p de S. cerevisiae pelo RNA da região ITS2, conforme observado em ensaios de interação utilizando UV-crosslinking. De fato, uma análise funcional realizada por primer extension comprovou que ocorre um bloqueio no processamento da região espaçadora ITS2 na ausência de Nip7p. Nip7p interage com várias proteínas do complexo pré-60S, entre as quais Nop8p e Nop53p, ambas associadas ao processamento do pré-27S. Embora os ensaios de co-purificação tenham confirmado a interação com as proteínas do complexo H/ACA box, deficiência em Nip7p não afeta a pseudo-uridinilação do rRNA. O duplo-híbrido realizado com a ortóloga humana de Nip7p, HsNip7, revelou interações com FTSJ3 e com a proteína SUMO-2. A interação direta de HsNip7 com estas proteínas foi confirmada por ensaios in vitro. HsNip7 e FTSJ3 colocalizaram na região nucleolar de células HEK293. FTSJ3 é uma proteína não caracterizada que possui o domínio FtsJ, descrito inicialmente para rRNA metiltransferases de procariotos. Além disso, FTSJ3 apresenta similaridade de sequência à proteína Spb1p de levedura, cuja função na metilação do rRNA 25S na posição Gm2922 já foi estabelecida. Embora a Nip7p não interaja com a Spb1p, estes dados indicam que FTSJ3 deve ser a ortóloga humana da Spb1p. As proteínas SUMO estão envolvidas na modificação pós-traducional (sumoylation) que regula a localização subcelular de proteínas. Em levedura, a provável ortóloga de SUMO, Smt3p, foi descrita na partícula pré-60S, portanto a interação HsNip7-SUMO-2 pode ser específica. Estes dados sugerem que as proteínas atuem no mesmo complexo da formação da subunidade 60S também em células humanas
Abstract: Ribosome biogenesis is conserved throughout eukaryotes and takes place in the nucleolus, a specialized nuclear compartment where the rRNA precursors are transcribed. More than 170 trans-acting factors coordinately interact to generate the mature rRNAs. Among the proteins identified in the pre-60S particle in Saccharomyces cerevisiae is Nip7p. Highly conserved Nip7p orthologues are found in all eukaryotes and Archaea. The analysis of Nip7p sequence reveals a conserved C-terminal domain named PUA, also found in a number of RNA-interacting proteins. In this work, we performed structural and functional analysis to investigate Nip7p molecular role on rRNA processing and modification. The structure of Pyrococcus abyssi Nip7p ortholog, PaNip7, was solved using X-ray diffraction data to 1,8Å resolution. Structural analysis followed by in vitro assays confirmed the involvement of PUA domain in RNA interaction. S. cerevisiae Nip7p and its archaeal and human counterparts show preference for binding uridine-rich sequences, indicating conserved functional features among the orthologues. The preference for uridine can explain the higher affinity of S. cerevisiae Nip7p for ITS2 sequence, as observed by UV-crosslinking assays. Consistently, functional analysis revealed pre-rRNA processing in the ITS2 region is seriously impaired. Yeast two-hybrid analysis confirmed by pull down assays revealed Nip7p interacts with Nop8p and Nop53p, two nucleolar proteins involved in pre-27S processing and components of pre-60S particle. Although yeast two-hybrid and pull down assays indicated that Nip7p interacts with H/ACA box core proteins, pseudouridylation is not affected under conditions of Nip7p depletion. In addition, yeast two-hybrid analysis confirmed by GST-pull down revealed HsNip7 interaction with FTSJ3 and SUMO-2. Both HsNip7 and FTSJ3 showed nucleolar subcellular localization in HEK293 cells. FTSJ3 is an uncharacterized protein containing the FtsJ domain, initially described in prokaryotic rRNA methyl-transferases. FTSJ3 shows sequence similarity to yeast Spb1p, an rRNA methyl-transferase involved in methylation of Gm2922, indicating that FTSJ3 may be the human orthologue of Spb1p. Sumoylation is a post-transcriptional covalent modification involved in regulation of protein subcellular localization. Putative yeast orthologues of SUMO, such as Smt3p, have been described in the pre-60S ribosomal particle, suggesting that SUMO-2 might play a specific role in 60S subunit biogenesis
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular

Presented here is a comprehensive computational survey of evolutionarily conserved secondary structure motifs in the genomic RNAs of the family Flaviviridae. This virus family consists of the three genera Flavivirus, Pestivirus and Hepacivirus and the group of GB virus C/hepatitis G virus with a currently uncertain taxonomic classification. Based on the control of replication and translation, two subgroups were considered separately: the genus Flavivirus, with its type I cap structure at the 5′ untranslated region (UTR) and a highly structured 3′ UTR, and the remaining three groups, which exhibit translation control by means of an internal ribosomal entry site (IRES) in the 5′ UTR and a much shorter less-structured 3′ UTR. The main findings of this survey are strong hints for the possibility of genome cyclization in hepatitis C virus and GB virus C/hepatitis G virus in addition to the flaviviruses; a surprisingly large number of conserved RNA motifs in the coding regions; and a lower level of detailed structural conservation in the IRES and 3′ UTR motifs than reported in the literature. An electronic atlas organizes the information on the more than 150 conserved, and therefore putatively functional, RNA secondary structure elements.

The genomes of RNA viruses often carry conserved RNA structures that perform vital functions during the life cycle of the virus. Such structures can be detected using a combination of structure prediction and co-variation analysis. Here we present results from pilot studies on a variety of viral families performed during bioinformatics computer lab courses in past years.

In contrast to the fairly reliable and complete annotation of the protein coding genes in the human genome, comparable information is lacking for noncoding RNAs (ncRNAs). We present a comparative screen of vertebrate genomes for structural noncoding RNAs, which evaluates conserved genomic DNA sequences for signatures of structural conservation of base-pairing patterns and exceptional thermodynamic stability. We predict more than 30,000 structured RNA elements in the human genome, almost 1,000 of which are conserved across all vertebrates. Roughly a third are found in introns of known genes, a sixth are potential regulatory elements in untranslated regions of protein-coding mRNAs and about half are located far away from any known gene. Only a small fraction of these sequences has been described previously. A comparison with recent tiling array data shows that more than 40% of the predicted structured RNAs overlap with experimentally detected sites of transcription. The widespread conservation of secondary structure points to a large number of functional ncRNAs and cis-acting mRNA structures in the human genome.

In recent years it has become evident that functional RNAs in living organisms are not just curious remnants from a primoridal RNA world but an ubiquitous phenomenon complementing protein enzyme based activity. Functional RNAs, just like proteins, depend in many cases upon their well-defined and evolutionarily conserved three-dimensional structure. In contrast to protein folds, however, RNA molecules have a biophysically important coarse-grained representation: their secondary structure. At this level of resolution at least, RNA structures can be efficiently predicted given only the sequence information. As a consequence, computational studies of RNA routinely incorporate structural information explicitly. RNA secondary structure prediction has proven useful in diverse fields ranging from theoretical models of sequence evolution and biopolymer folding, to genome analysis and even the design biotechnologically or pharmaceutically useful molecules.

Ribosomes are multicomponent macromolecular particles and are essential for the survival of cells in all organisms. The ribosome's universal function is to catalyze polypeptide synthesis through translation of mRNA transcripts. Ribosomes from Escherichia coli, eubacterial organisms, have a sedimentation coefficient of 70S and are composed of 30S and 50S ribonucleoprotein subunits. The small ribosomal subunit is an assembly of 21 different proteins and a 16S ribosomal RNA. Within the 16S rRNA there are a few short stretches of universally conserved sequences spanning positions 517-533, 1394-1408, and 1492-1506. Clear functions for these sequence zones have not yet been assigned. Here I report a kinetic analysis of these highly conserved regions in the 16S rRNA and within the 30S ribosomal subunits. Binding affinity was measured in experiments that were based on protection from nuclease 51 digestion of short oligodeoxynucleotides hybridized to the designated regions. DNAs hybridized to regions 1400 and 1500 show significant differences in the apparent dissociation constants when measured in 30S particles as opposed to those found for 16S rRNA. Region 525 showed no difference in kinetic behavior. To further elucidate the functional and structural role played by the region centered about C1400 in 16S rRNA, a four nucleotide deletion was constructed within this region. The deletion was introduced by direct RNA manipulation using DNA/RNA hybridization, RNase H digestions, and ligation of the correct RNA fragments with T4 RNA ligase. I improved ligation efficiency of large RNA molecules by including a connector looped short DNA oligomer. Recycling products through phenyl boronate agarose (PBA-30) column also improved the efficiency of ligation. The mutagenized 16S rRNA fully reassembles into 30 particles and the altered 30S subunit possesses all of the normal ribosomal proteins. Altered ribosomes were functional in in vitro translation of MS2 mRNA. The altered ribosomes have lower translational activity relative to controls. Here I present indirect evidence suggesting that the decrease in the synthesis of MS2 coat proteins is the result of premature termination. The altered 16S RNA in ribosomes had an apparent dissociation constants with DNA probes comparable to those found for normal 16S rRNA. This suggest that the RNA is less flexible in the particle relative to normal 30S subunits. The deletion at 1400 did not have any effect on the physical properties of the 1500 region, as measured by DNA hybridization. A minor, but significant, effect on the 525 region was observed. A possible RNA/RNA interaction within the 30S particle is proposed to account for this observation.
Graduation date: 1992