561 research outputs found
Positive and Negative Regulatory Elements in the HIV-1 5'UTR Control Specific Recognition by Gag
No full textBiological Sciences (The Ohio State University Denman Undergraduate Research Forum)The 5ʹ untranslated region (5ʹUTR) of the human immunodeficiency virus type 1 (HIV-1) genomic RNA (gRNA) contains a structured RNA element (termed Psi) that is specifically recognized by the HIV-1 Gag polyprotein, ensuring that two strands of gRNA are packaged into newly assembled virions. However, the mechanism by which Gag recognizes gRNA over other cellular RNAs and spliced viral RNAs is not well understood. A recent study suggested that a negative regulatory element upstream of Psi reduces high-affinity Gag binding, and a positive regulatory element downstream of Psi counteracts the upstream element and restores high-affinity binding. The aim of this study is to determine how these elements affect the specificity and mode of Gag binding. Using a fluorescence anisotropy-based salt-titration binding assay, the electrostatic and nonelectrostatic (i.e., specific) components of binding were measured. We have previously shown that Gag interacts with a 109-nucleotide (nt) Psi RNA construct that lacks the putative regulatory elements with high specificity and relatively few electrostatic interactions. Using a 356-nt RNA construct that includes the negative regulatory element in addition to Psi, we observed a loss in Gag binding specificity and an increase in electrostatic interactions. Interestingly, a 400-nt construct that contains the positive and negative elements flanking Psi restored highly specific binding and reduced the electrostatic interactions made with the RNA. Furthermore, a construct wherein the 40-nt positive regulatory element was appended to Psi, demonstrated the same specificity as Psi alone. Taken together, these data are consistent with a mechanism whereby the negative and positive regulatory elements flanking Psi modulate Gag binding mode and specificity.Undergraduate Education Summer Research FellowshipArts & Sciences Undergraduate Research ScholarshipSOLAR Research GrantNo embargoAcademic Major: Biochemistr
Discriminator Base is a Critical Recognition Element for Trypanosoma brucei Ala-tRNAPro Editing Domains
No full textProlyl-tRNA synthetases (ProRSs) mischarge Ala onto cognate tRNAPro. In bacteria, this error can be corrected by a cis-editing domain appended to the synthetase (INS) or by a homologous freestanding trans-editing domain, ProXp-ala. Trypanosoma brucei (Tb) ProRS encodes an appended ProXp-ala domain and a unique free-standing INS homolog—MCP3 (multi-tRNA synthetase complex 3 protein). We hypothesize that Tb may have two Ala-tRNAPro editing domains due to its unusual metabolism. In the procylic form, the main carbon source is proline, with alanine produced as a metabolic by-product. With high intracellular concentrations of alanine, robust editing mechanisms may be needed to avoid misincorporation of Ala at Pro codons. Free-standing Homo sapiens (Hs) ProXp-ala recognizes nucleotides in the tRNA acceptor stem for optimal function. In contrast, the ProRS-appended E. coli INS domain relies exclusively on ProRS anticodon recognition. Here, we probed the acceptor stem specificity of Tb Ala-tRNAPro editing domains. Ala-tRNAPro deacylation assays revealed that the activities of ProRS-appended Tb ProXp-ala, as well as free-standing ProXp-ala and MCP3, are significantly decreased upon mutation of the discriminator base (C73A), with more relaxed recognition of the first base pair (G1:C72). Surprisingly, MCP3 deacylated the triple mutant (G1C:C72G, C73A) Ala-tRNAPro better than the C73A single mutant, and displayed robust deacylation of G1:C72, A73-containing Ala-tRNAAla in vitro. Thus, the context of the discriminator base and the Ala moiety appear to be important determinants for this editing domain. As MCP3 is not encoded in the human genome, results of these studies may have implications for new therapeutic strategies.The Center for RNA Biology (OSU)No embargoAcademic Major: Microbiolog
HIV-1 Primer Binding Site:Lysyl-tRNA Synthetase Interaction Affinity Diminishes Upon tRNA Primer Annealing and Extension
No full textBiological Sciences: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)The retrovirus, human immunodeficiency virus type 1 (HIV-1), possesses a positive sense RNA genome (gRNA) that is reverse transcribed into proviral DNA upon infection. In order for reverse transcription to occur, HIV-1 co-opts cellular tRNALys3, whose 3 ́ 18 nucleotides are perfectly complimentary to a region with the gRNA, to serve as the primer. tRNALys3 is selectively packaged into virions through its interaction with the cellular enzyme lysyl-tRNA synthetase (LysRS), which in turn interacts with the viral protein responsible for orchestrating virus assembly, Gag. However, the mechanism of tRNALys3 transfer from the packaged LysRS to the primer-binding site (PBS) remains incompletely understood. The PBS is harbored in the 5 ́ untranslated region (5 ́UTR) of the gRNA, a highly conserved segment of the HIV-1 genome. We have recently found that a U-rich stem loop immediately upstream of the PBS mimics the anticodon loop nucleotides of tRNALys3, a critical LysRS recognition element. This tRNA-like element (TLE) specifically binds to LysRS, and can competitively displace tRNALys3 from the synthetase. Furthermore, small angle X-ray scattering analysis revealed that the whole PBS domain (PBS105) mimics the overall 3D shape of tRNA. Overall, these data suggest a mechanism where structural and functional tRNA mimicry by the TLE in the PBS domain facilitate primer release from LysRS and targeting to the 18 nucleotide PBS.
An observation from the structural analysis was that both apoPBS and PBS annealed to a DNA oligonucleotide corresponding to the 18 complementary nucleotides in tRNALys3 (antiPBS18) mimicked the overall tRNA shape to a similar degree. In order to further investigate the function of the PBS/TLE domain, we performed a fluorescence anisotropy-based binding study examining LysRS interactions with the PBS domain in various functionally relevant states. We find LysRS has similar affinities for both the apoPBS105 and PBS105:antiPBS18 complex, confirming the SAXS structure indicating both complexes mimic tRNA shape. In order to investigate if the additional tRNA-gRNA contacts outside of the 18 nucleotides of complementarity affected the LysRS interaction, we tested PBS105:primer complexes containing full-length and 3 ́-half tRNAs, finding that PBS:tRNA primer complexes displayed reduced affinities for LysRS under certain conditions. Also, when progressively extended antiPBS18 primers were annealed to PBS105, mimicking the initial steps of reverse transcription, we observed a concomitant drop in LysRS affinity. These data further elucidate the role that LysRS plays in the evolution of the reverse transcription initiation complex.A five-year embargo was granted for this item
Phosphorylation State Modulates Role of SARS-CoV-2 Nucleocapsid Protein in Viral Replication
No full textSARS-CoV-2, the causative agent of COVID-19, is a positive-sense single-stranded betacoronavirus. Significant progress has been made in understanding the SARS-CoV-2 lifecycle. The Nucleocapsid protein (N protein) is responsible for viral RNA (vRNA) packaging, plays a vital role in replication and transcription, and is associated with viral replication-transcription complexes. N protein contains a conserved Ser-Arg (SR)-rich disordered linker between its two structured domains, which is a known site of phosphorylation by multiple host kinases; modification is proposed to regulate its function during infection. Phosphorylated N protein has been proposed to participate in viral transcription while non-phosphorylated N protein packages gRNA into new virions. However, little is known about N protein binding specificity to nucleic acids, and how it is modulated by phosphorylation. The 5' untranslated region (UTR) of vRNA has conserved secondary structures that are necessary for vRNA replication. We used RNA constructs derived from the SARS-CoV-2 5' UTR containing either stem loops (SLs) 1-4, SL5, or SL1-5, as well as HIV-1 derived RNAs. We also prepared phosphomimetic N protein mutants with 3 Ser/Thr to Asp mutations in the N-terminal region of the SR linker (3xD1) or the C-terminal region (3xD2), as well as a variant with all 6 positions mutated (6xD). Fluorescence anisotropy (FA) direct binding experiments and FA salt-titration binding assays were used to compare salt dependence and binding specificity among non-phosphorylated wild-type and mutant proteins. Trypsin time course digestion was utilized to analyze structural differences between wild-type and mutant proteins as well. Direct binding assays showed that phosphomimetic mutants and non-phosphorylated N protein have high-affinity binding (low nM Kd’s) to all RNAs tested. The non-phosphorylated N protein bound with similar specificity to all SARS2 and HIV-1 RNAs tested with a strong non-electrostatic component. The 6xD phosphomimetic N protein mutant displayed high selectivity for SARS2 RNAs and bound primarily electrostatically with HIV-1 RNAs. The location of the phosphomimetic mutations determined whether binding was primarily electrostatic (3xD1) or hydrophobic in nature (3xD2). Overall, this data supports the conclusion that the location of phosphorylation within the SR linker of SARS-CoV-2 N protein modulates its structure and binding to RNA.National Institute of Health (NIH)A three-year embargo was granted for this item.Academic Major: Biochemistr
Purification and in vitro characterization of Trypanosoma brucei prolyl-tRNA synthetase
No full textHousekeeping proteins include a broad variety of enzymes that complete the basic tasks
necessary for cell survival. Among this group, aminoacyl-tRNA synthetases (aaRSs) function to
attach the correct amino acid to the cognate tRNA substrate in a reaction known as
aminoacylation. The aminoacyl-tRNA is delivered to the ribosome by an elongation factor,
where it participates in protein synthesis. While catalysis of the aminoacylation reaction seems
straightforward, aaRSs must sample similarly sized amino acids to find a match for the protein
active site. Amino acids with the same or smaller molecular volume can fit into the active site
and become mistakenly charged to noncognate tRNA. Thus, the cell uses proofreading
mechanisms to ensure that only cognate aminoacyl-tRNAs arrive at the ribosome. In the context
of charging L-proline to tRNAPro, prolyl-tRNA synthetase (ProRS) frequently mischarges
alanine and cysteine and generates Ala-tRNAPro and Cys-tRNA
Pro. The bacterial Escherichia coli
enzyme, for example, relies on an insertion (INS) domain in its ProRS architecture to hydrolyze
mischarged Ala-tRNAPro species. Other prokaryotes such as Caulobacter crescentus, lack this
embedded domain but encode a homologous, free-standing editing factor, ProXp-ala, for the
same function. Beyond prokarya, however, understanding of eukaryotic editing processes
remains limited. Homo sapiens and other higher eukaryotes also possess a free-standing ProXp ala domain for editing. Bioinformatic analyses have revealed lower eukaryotes with a ProRS
architecture reminiscent of E. coli. These eukaryotes encode a canonical ProRS with a fused N terminal ProXp-ala domain. Because many organisms with this understudied ProRS structure are
parasites such as Plasmodium falciparum, Leishmania tarentolae, and Trypanosoma brucei,
potential nuances in tRNA recognition and editing mechanism hold promise for drug targeting.
Inhibition of Trypanosoma brucei (Tb) ProRS editing, for example, may provide a cure for
African Sleeping Sickness while avoiding the human system entirely. In this work, we attempted
to overexpress and purify full-length Tb ProRS for the first time in E. coli. While purification of
the full-length construct was challenging due to poor solubility and low yields, the individual
ProXp-ala and ΔProXp-Ala ProRS constructs were generated via SLIM-PCR, expressed, and
purified with appreciable yield. The Tb ΔProXp-Ala ProRS enzyme activity was probed via
aminoacylation reactions. Pure Tb tRNAPro was generated by in vitro transcription and used in
aminoacylation reactions with the Tb ProRS domain to generate Pro-tRNAPro. Flexizyme
charging was used to generate Tb Ala-tRNAPro. Deacylation assays were performed with the
purified Tb ProXp-ala domain to provide preliminary kinetic data demonstrating the editing
capabilities of Tb ProRS. Although these assays provide no explanation for the selective pressure
driving this fused architecture, they do provide preliminary evidence that Tb ProXp-ala is an
Ala-tRNAPro deacylase in vitro. Comparison to other trans editing factors revealed that the
isolated Tb ProXp-ala domain is a weaker deacylase; however, the presence of the fused ProRS
domain may be required for more robust deacylation activity and future work will address this
hypothesis. Overall, we have demonstrated that the isolated Tb ProRS domains—both behave as
predicted by bioinformatic analysis and provide further impetus for the purification of the full-length protein.Center for RNA BiologyNo embargoAcademic Major: Biochemistr
Role of the Bifunctional Aminoacyl-tRNA Synthetase EPRS in Human Disease
No full textAminoacyl-tRNA synthetases (AARS) are a class of enzymes that catalyze the charging of tRNAs with cognate amino acids, a critical step that contributes to the fidelity of protein synthesis. Many AARSs also possess noncanonical functions such as regulation of apoptosis, mRNA translation, and RNA splicing. Some AARSs have evolved new domains with no apparent connection to their charging functions. For example, WHEP domains were originally identified in tryptophanyl-tRNA synthetase (WRS), histidyl-tRNA synthetase (HRS), and glutamyl-prolyl-tRNA synthetase (EPRS). EPRS is a unique bifunctional AARS, found only in higher eukaryotes, and consists of glutamyl-tRNA synthetase (ERS) and prolyl-tRNA synthetase (PRS) joined by a non-catalytic linker containing three WHEP domains in humans. Two compound heterozygous point mutations within human ERS (P14R and E205G) have been identified in the genomes of two patients with type 1 diabetes and bone disease. However, the mechanism by which these mutations contribute to disease is unknown. Our goal is to determine whether the point mutations affect the canonical catalytic activity of EPRS responsible for tRNA charging or noncanonical functions. Both P14 and E205 are highly conserved residues located in the GST and catalytic domain, respectively. An ERS variant appended to 2.5 WHEP domains (ERS 2.5W) has been purified and shown to display robust tRNA binding and aminoacylation activity in vitro. The P14R and E205G single mutants display the same binding affinity for tRNAGlu as WT ERS 2.5W, suggesting that the observed defect is at the catalytic step. Whereas the ERS 2.5W P14R mutant has near wild-type (WT) aminoacylation activity, the ERS 2.5W E205G variant has a severe aminoacylation defect. Both mutations, however, lead to reduced amino acid activation. Together with a collaborator, we are currently characterizing the effect of these two mutations on cell proliferation and the integrated stress response. Taken together, this work has important implications for the understanding of AARS-related human disease mechanisms and development of new therapeutics.College of Arts & SciencesOffice of Undergraduate Research & Creative InquiryNo embargoAcademic Major: Biochemistr
Probing the Role of Gag in Regulation of Reverse Transcription
No full textThe HIV-1 nucleocapsid (NC) domain of Gag has both specific and more general nucleic acid (NA) binding properties. NC acts specifically to recognize and package unspliced viral RNA and as a general chaperone to facilitate reverse transcription by annealing/ aggregating NAs via its highly positive character and by destabilizing NA secondary structure via its two CCHC zinc finger (ZF) motifs. Interestingly, it has recently been reported that ZF deletion or mutation to CCCC results in production of virions containing DNA instead of RNA, rendering them noninfectious. Thus, an additional role of NC is to prevent premature reverse transcription from occurring before or during assembly. Here, we probe the in vitro NA binding and chaperone properties of Gag variants containing the same ZF mutations or deletions tested in the cell-based assays. Fluorescence anisotropy equilibrium binding measurements reveal that mutation or deletion of both ZFs results in a modest reduction in binding to the ψ SL3 stem-loop relative to WT Gag, whereas binding to nonspecific single-stranded NAs is largely unaffected. Similarly, Gag’s ability to aggregate NAs or facilitate tRNALys,3 annealing to the primer-binding site, two functions of Gag during viral assembly, was only moderately affected upon ZF mutation or deletion. A time-resolved fluorescence resonance energy transfer assay was used to monitor hairpin stem opening of a DNA hairpin construct. Surprisingly, single CCCC and ZF deletion variants appeared to be more effective at opening the TAR hairpin than WT Gag, and Gag variants in which both zinc fingers were mutated or deleted showed even greater duplex destabilization capability than the single ZF variants. Previous studies with the freestanding NC domain of Gag indicated that NC’s duplex destabilization activity depended on the ZF structures. Our new results suggest that in the context of Gag, disruption of the ZF leads to an increased ability to disrupt nucleic acid secondary structure and we explore possible mechanisms by which this increased capability may cause premature reverse transcription.The Ohio State UniversityThe National Science FoundationThe National Institute of HealthNo embarg
Retroviral-RNA Structure and Function: Investigating the role of aminoacyl-tRNA synthetases and retroviral-RNA structural elements in the initiation of reverse transcription
No full textMost human retroviruses are not known to be pathogenic. In fact, the only two retroviruses that have been shown to cause human disease are human immunodeficiency virus (HIV), and human T-cell lymphotropic virus type 1 (HTLV-1)—the causes of acquired immunodeficiency syndrome (AIDS), and adult T-cell leukemia/lymphoma (ATL) among other diseases, respectively. The hallmark of a retrovirus is reverse transcription (RT), the process through the which RNA genome is copied from ssRNA to dsDNA. However, for RT to initiate, a primer must specifically anneal to the primer binding site (PBS) in the 5′ untranslated region (5′UTR) of the retroviral genome. HIV-1 and HTLV-1 use tRNALys3 and tRNAPro to primer RT respectively. Our lab has demonstrated that human lysyl-tRNA synthetase (LysRS) facilitates priming in HIV-1 by binding a tRNA-like-element (TLE) near the PBS, localizing the tRNALys3 primer. As the primer for HTLV-1 RT is tRNAPro, we hypothesized that this virus may employ a similar mechanism involving human glutamyl-prolyl-tRNA synthetase (EPRS) binding to a TLE. Chapter 1 of this thesis will discuss the optimization of RNA probing experiments to study these retroviral RNAs, improvements made to the processing of RNA probing data analyzed by capillary electrophoresis, and a user friendly tool under development to facilitate this processing. Chapter 2 will discuss the probing of the LysRS:HIV-1 interaction, and small angle X-ray scattering studies aimed to understand more about the structure and function of the HIV-1 5’UTR. Finally, Chapter 3 will discuss investigations of the putative interaction between elements within the HTLV-1 5′UTR and EPRS. Using a wide variety of biochemical techniques, these studies aspire to gain information about these viral processes that could eventually aid in the production of therapeutic agents.No embargoAcademic Major: Biochemistr
Investigating the effect of phosphomimetic mutations of SARS-CoV-2 nucleocapsid protein on viral RNA binding
No full textInnovations in Medicine (The Ohio State University Denman Undergraduate Research Forum)SARS-CoV-2, the causative agent of COVID-19, is a positive-sense single-stranded betacoronavirus. Significant progress has been made in understanding the SARS-CoV-2 lifecycle. The Nucleocapsid protein (Np) is responsible for viral RNA (vRNA) packaging, plays a vital role in replication and transcription and is associated with viral replication-transcription complexes. Np contains a conserved Ser-Arg (SR)-rich disordered linker between its two structured domains, which is a known site of phosphorylation by multiple host kinases; modification is proposed to regulate its function during infection. Phosphorylated Np has been proposed to participate in viral transcription while non-phosphorylated Np packages gRNA into new virions. However, little is known about Np binding specificity to nucleic acids, and how it is modulated by phosphorylation. The 5’ untranslated region (UTR) of vRNA has conserved secondary structures that are necessary for vRNA replication. We used RNA constructs derived from the SARS-CoV-2 5’UTR containing either stem loops (SLs) 1-4, SL5, or SL1-5, as well as HIV-1 derived RNAs. We also prepared phosphomimetic Np mutants with 3 Ser/Thr to Asp mutations in the N-terminal region of the SR linker (3D1) or the C-terminal region (3D2), as well as a variant with all 6 positions mutated (6D). Direct binding experiments using fluorescence anisotropy (FA) and FA salt-titration binding assays were used to compare salt dependence and binding specificity among non-phosphorylated wild-type and mutant proteins. Trypsin time course digestion was utilized to analyze structural differences between wild-type and mutant proteins as well. Direct binding assays showed that phosphomimetic mutants and non-phosphorylated Np have high-affinity binding (low nM Kd’s) to all RNAs tested. The non-phosphorylated Np protein bound with similar specificity to all SARS and HIV-1 RNAs tested with a strong non-electrostatic component. The 6D phosphomimetic Np mutant displayed high selectivity for SARS RNAs and bound primarily electrostatically with HIV-1 RNAs. The location of the phosphomimetic mutations determined whether binding was primarily electrostatic (3D1) or hydrophobic in nature (3D2). Overall, this data supports the conclusion that the location of phosphorylation within the SR linker of SARS-CoV-2 Np modulates its structure and binding to RNA.A three-year embargo was granted for this item.Academic Major: Biochemistr
Novel Tetrahelical Monomolecular Architecture of Nucleic Acids Probed by NMR
No full textWe recently described a tetrahelical monomolecular architecture of nucleic acids that employs G (guanine)-quartets as a basic structural element. The monomeric units of the architecture, GGGTGGGTGGGTGGG (G3T) or its RNA analog, g3u, are folded in an intramolecular quadruplex with three G-quartets connected to each other by chainreversal or propeller loops. In this architecture, the G3T or g3u monomers are stacked on each other forming an extraordinarily stable uninterrupted polymer. Based on thermodynamic and melting studies, we propose that the tetrahelical architecture consists of n G3T domains, (G3T)n, wherein the terminal G3 segments of adjacent G3T domains form G6-segments. Here, we employ proton NMR to probe the solution structure of (G3T)2. The NMR data are consistent with formation of an uninterrupted homopolymer consisting of repetitive G-tetrads with strong structural symmetry. Specific guanine-toinosine and alternation of D2O concentration also presented detailed analysis of the (G3T)2 structure. The tetrahelical architecture has strong potential in DNA nanotechnology as a programmable alternative to a DNA duplex.The Scholarship for Research DistinctionA three-year embargo was granted for this item.Academic Major: Biomedical Engineerin
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