Browsing by Subject "Reverse transcriptase"

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The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants
(2010-08) Syrett, Heather Angel; Ellington, Andrew D.; Kitto, George B.; Willets, Katherine A.; Iyer, Vishwanath R.; Yin, Yuhui W.
The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun.
Conformational dynamics plays a significant role in HIV reverse transcriptase resistance and substrate selection
(2012-12) Nguyen, Virginia Myanh; Johnson, Kenneth A. (Kenneth Allen)
Human immunodeficiency virus reverse transcriptase (HIV RT) is a virally encoded polymerase responsible for replicating the HIV genome. Most HIV treatments include nucleotide RT inhibitors (NRTIs) which inhibit HIV RT replication by serving as a substrate for the polymerase reaction but then blocks subsequent polymerization after incorporation. However, resistance to these NRTIs may occur through specific mutations in HIV RT that increase the discrimination of HIV RT for natural nucleotides over NRTIs. The role of enzyme conformational dynamics in specificity and substrate selection was studied using transient kinetic methods on HIV RT enzymes that have been site-specifically labeled with a conformationally sensitive fluorophore, to measure the rates of binding and catalysis. First, HIV RT with the mutation of lysine to arginine at the residue position 65 (K65R) was examined for its resistance against the NRTI tenofovir diphosphate (TFV), an acyclic deoxyadenosine triphosphate (dATP) analog. It was found that HIV RT K65R resistance to TFV was achieved through decreased rates of catalysis and increased rates of dissociation for TFV over dATP when compared with the kinetics of wild-type HIV RT. Moreover, global fitting analysis confirmed a mechanism where a large conformational change, after initial ground state binding of the substrate, contributed significantly to enzyme specificity. This led to our investigation of the molecular basis for enzyme specificity using HIV RT as a model system. Again, transient kinetic methods were applied with the addition of molecular dynamics simulations. The simulated results were substantiated by the corroborating experimental results. It was found that a substrate-induced conformational change in the transition of HIV RT from an open nucleotide-bound state to a closed nucleotide-bound state was the major determinant in enzyme specificity. The molecular basis for substrate selection resulted from the molecular alignments of the substrate in the active-site, which induced the conformational change. When the correct nucleotide was bound, optimal molecular interactions in the active-site yielded a stably closed complex, which promoted nucleotide incorporation. In contrast, when an incorrect nucleotide was bound, the molecular interactions at the active-site were not ideal, which yielded an unstable closed complex, which promoted substrate dissociation rather than incorporation.
Directed evolution of T7 RNA polymerase variants using an 'autogene'
(2004-08) Chelliserrykattil, Jijumon Pavithran, 1974-; Ellington, Andrew D.
Natural enzymes, when used in biotechnological applications are often found not well suited for the tasks. Enzyme properties can now be improved by rational design or by directed evolution to produce useful biocatalysts. The work described in this thesis is mainly focused towards developing a directed evolution method to evolve RNA polymerases with novel properties, such as the ability to use modified nucleotides as substrates. A variety of modified nucleotides can be imagined that can impart unique characteristics to RNA molecules into which they are incorporated. Modified nucleotides improve the functionality of RNA, but more importantly, they increase the stability of RNA towards nucleases thus lending the RNA amenable to various biotechnological applications. Modified RNA transcripts will also find applications in the in vitro selection of aptamers and ribozymes. We have developed a directed evolution method for the isolation of RNA polymerase variants with altered promoter specificities and novel substrate specificities vii using a construct called an “autogene”. The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNA polymerase) is being used for the directed evolution studies. In short, a library of T7 RNA polymerase variants was made by randomizing the gene of T7 RNA polymerase at amino acid positions that are important for a desired activity (for e.g.: altered promoter recognition). This gene was then cloned downstream of a T7 promoter, generating a so-called autogene library. Following transformation to E.coli, those polymerase variants that best recognized their adjacent promoter self-amplified both their mRNAs and themselves in vivo. The variant mRNAs extracted from the population as a whole will be roughly represented according to the activities of their corresponding variant polymerases. Following reverse transcription and PCR amplification in vitro, the most abundant polymerase genes were carried into subsequent rounds of selection. The method allows large (103 -106 ) polymerase libraries to be efficiently searched for their promoter recognition ability and fidelity. Autogene selection was subsequently modified with a reporter gene and used to screen polymerase variants that can incorporate modified nucleotides into the RNA backbone. We have successfully evolved a novel T7 RNA polymerase variant that transcribes 2’-O-methyl RNA. Other selections can also be envisioned using the autogene system to discover new polymerases with novel abilities. The polymerases thus evolved were used to construct modified RNA libraries to be used in in vitro selection of modified ribozymes.
Kinetic analysis of HIV-1 reverse transcriptase in the presence of non-nucleoside inhibitors
(2004) Wang, Louise Zhiying, 1973-; Johnson, Kenneth A. (Kenneth Allen)
Current treatment for human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS) delays the conversion of infected helper T cells to monoclonal malignant cells but does not eradicate HIV. A novel non-nucleoside inhibitor, CZ-1 (2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5- hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2- naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt), was designed to bind at an unconventional site on HIV type 1 reverse transcriptase (RT) (1). I examined the effect of CZ-1 on the kinetic parameters governing the single nucleotide polymerization. CZ-1 decreased the amplitude of the reaction as previously shown for other non-nucleoside inhibitors due to the slow equilibration of the inhibitor with RT. CZ-1 also weakened the apparent DNA binding affinity. Likewise, DNA weakened the apparent CZ-1 binding affinity. In contrast, CZ-1 did not affect the Kd and the maximum incorporation rate of the incoming nucleotide. We therefore conclude that CZ-1 represents a new class of NNRTIs distinct from Nevirapine and related NNRTIs. CZ-1 can bind to RT in both the absence and presence of DNA. In the ternary enzyme-DNA-CZ-1 complex, the binding of DNA is weakened and incorporation of the next nucleotide onto the primer is inhibited. A possible mode of inhibition for CZ-1 is the distortion of RT conformation and the consequent misalignment of DNA at the active site. Unexpectedly, CZ-1 also inhibited human mitochondrial DNA polymerase (pol γ), although I did not see evidence of nonspecific inhibition via self-aggregation or intercalation with DNA. The second part of my project aimed to characterize a non-nucleoside drug-resistant mutant form of HIV-1 RT, containing a single amino acid substitution at position 103 from lysine to asparagine. Three non-nucleoside RT inhibitors, CZ-1, HBY 097, and α-APA, exhibited similar inhibition kinetics on this mutant as on the wild type RT. The third part of my project determined that HIV RT exhibited similar polymerization kinetics whether utilizing an RNA template or DNA template. This validation suggests that the measured kinetic parameters were similar for RNA-directed minus-strand DNA synthesis and DNA-directed plus-strand DNA synthesis.
Mechanism of DNA target site recognition by group II introns TeI3c and GsI-IIC and splicing activity of GsI-IIC reverse transcriptase
(2016-12) Kang, Sean Yoon-Seo; Lambowitz, Alan
Mobile group II introns are self-catalytic ribozymes found in bacteria and eukaryotic organelles. They can mobilize within the genomes by retrohoming, which involves RNA-catalyzed splicing followed by the excised intron reverse splicing into a target site. Both RNA splicing and retrohoming are facilitated by an intron-encoded reverse transcriptase (RT). Mobile group II introns are of interest as evolutionary ancestors of spliceosomal introns in higher organisms, for their use as bacterial gene targeting vectors known as targetrons, and as a source of thermostable group II intron reverse transcriptases (TGIRTs) for RNA-seq. The focus of this master’s thesis is on two thermophilic group II introns found in bacterial thermophiles: the subgroup IIB intron TeI3c and the subgroup IIC intron GsI-IIC. The TeI3c intron is known to rely on base pairing interaction between exon-binding site sequences 1/2 (EBS1/2), within the intron RNA, and intron-binding site sequences 1/2 (IBS1/2) in the 5’ exon of its target DNA, but it is not clear what targeting rules dictate one target sequence to be better or worse than others. I studied the targeting rules of TeI3c during retrohoming by using randomized libraries and next-generation sequencing followed by computational analysis of the sequence data. Understanding the targeting rules of TeI3c can be the important step in the development of thermostable targetron, which can be useful for metabolic engineering in the biofuel industry. Unlike TeI3c, which relies primarily on base pairing for DNA target recognition, the GsI-IIC intron recognizes a 5’-exon hairpin secondary structure of the target DNA. However, the secondary structure requirements of good targets have not been studied. I studied the secondary structure requirements during GsI-IIC retrohoming by using doped target libraries and next-generation sequencing to find conserved positions within a hairpin target site followed by mobility assays on different target sites with mutated conserved positions. Finally, I studied the forward splicing of GsI-IIC intron by comparing different hairpin target sites including the same mutated target sites tested for their mobility efficiency. These experiments address whether the 5’-exon hairpin structure is recognized similarly for RNA splicing and intron mobility.
Polar localization of a group II intron-encoded reverse transcriptase and its effect on retrohoming site distribution in the E. coli genome
(2007) Zhao, Junhua, 1976-; Lambowitz, Alan
The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein), which binds the intron RNA to promote RNA splicing and intron mobility. Mobility occurs by intron RNA reverse splicing directly into a DNA strand and reverse transcription by LtrA. I used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to the cellular poles in both Escherichia coli and L. lactis. This polar localization occurs with or without co-expression of the intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three non-overlapping LtrA subsegments, reflecting dependence on common redundant signals and/or protein physiochemical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. In E. coli, the Ll.LtrB intron inserts preferentially into the chromosomal ori and ter regions, which are pole localized during much of the cell cycle. Thus, the polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in these regions. I established a high throughput method using cellular array and automated fluorescence microscopy for screening transposon-induced mutants, and identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) in which disruptions result in increased proportion of cells having diffuse LtrA distribution. This altered localization is correlated with a more uniform distribution of Ll.LtrB insertion sites throughout the E. coli genome. Finally, I find that altered LtrA localization in all five disruptants is correlated with accumulation and more diffuse intracellular distribution of polyphosphate, and that a ppx disruptant, which also results in polyphosphate accumulation, shows similar LtrA mislocalization. These findings may reflect interaction between LtrA and intracellular polyphosphate. My findings support the hypothesis that the intracellular localization of LtrA is a major determinant of Ll.LtrB insertion site preference in the E. coli genome. Further, they show that alterations in polyphosphate metabolism can lead to protein mislocalization, and suggest that polyphosphate is an important factor affecting intracellular protein localization.
Single-molecule studies on the role of HIV-1 nucleocapsid protein/nucleic acid interaction in the viral replication cycle
(2007) Liu, Hsiao-Wei, 1974-; Barbara, P. F. (Paul F.), 1953-
The discovery of the crucial intermediates and pathway in the process of the reverse transcription was reported using single-molecule spectroscopy and related techniques including single-molecule fluorescence resonance energy transfer, fluorescence correlation spectroscopy and confocal imaging. Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. In this dissertation, the research focused on elucidating the mechanism of NC-facilitated TAR DNA/RNA annealing. The single molecule spectroscopic measurements reported that the crucial intermediates as well as the mechanistic insight into the annealing of TAR RNA with TAR DNA mediated by viral NC proteins. The data reveal that NC partially melted the secondary structure of TAR DNA (termed the "YTAR") as well as TAR RNA. In the subsequent studies, various short DNA oligonucleotdies were applied to anneal with the TAR to mimic the initial annealing steps. The data support that the YTAR serves as a nucleation center for the annealing to occur through the multiple sites along the TAR structure. Two major nucleation pathways were observed, which are the annealing through the 3'/5' termini, namely "zipper" pathway and the annealing through the hairpin loop region, namely "kissing" pathway. The annealing mechanism was further explored by performing the annealing of wild-type TAR DNA with wild-type TAR RNA in the presence of NC in vitro. The annealing kinetic data suggest that the nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the "Y" form associated with multiple NC molecules. This encounter complex leads to the multiple nucleation complexes, i.e. zipper or kissing intermediates. The data further indicate that although the two complementary strands nucleate at multiple sites, i.e. any single-strand region of TAR, the annealing of two TAR complements occurs through a common mechanism.
Structural investigations of the group II intron-encoded protein GsI-IIC
(2013-08) Rubinson, Max Edward; Lambowitz, Alan
Group II introns are a class of mobile ribozymes found in bacteria and eukaryotic organelles that self-splice from precursor RNAs. The resulting lariat intron RNA can then insert into new genomic DNA sites through a reverse splicing reaction. Collectively, this process of intron mobility is termed “retrohoming.” Mobile group II introns encode a reverse transcriptase (RT) that stabilizes the catalytically active form of the intron RNA for both the forward and reverse splicing reactions and also converts the integrated intron RNA into DNA. This work aims to elucidate the structure of bacterial group II intron-encoded RTs and ultimately determine how they function in intron mobility. Although efforts to crystallize group II introns RTs have been unsuccessful, small angle X-ray scattering studies in conjunction with homology modeling have provided new insights into the structure and function of these enzymes.
Structure-function analysis of a Group II Intron reverse transcriptase
(2022-05-09) Alvarado Torres, Jose Mario; Lambowitz, Alan; Russell, Rick
Mobile group II introns are ribozymes and mobile genetic elements found in all branches of life. They consist of a highly structured intron RNA that encodes a reverse transcriptase (RT). This RT is utilized during retrohoming to synthesize a full-length DNA copy (cDNA) of the intron RNA which is then integrated into new genome locations. One theory suggests that RTs evolved from an RNA Dependent RNA Polymerase (RDRP) that originated in the RNA World. These ancestral RDRPs served to propagate genetic material, and during the transition to DNA-based life, RDRPs evolved into RTs. Recent structural data for Geobacillus stearothermophilus Group II Intron RT (GsI-IIC RT) suggests a close evolutionary relationship between RDRPs and group II intron RTs, including a strikingly similar RT active site and regions that function in binding the template and primer. To further probe this evolutionary relationship rational mutations were introduced into GsI-IIC RT that affected rNTP incorporation ability. Group II introns have also potentially evolved characteristics like higher fidelity and processivity compared to other RTs due to stringent accuracy requirements when reverse transcribing the catalytically active and highly structured intron RNA. Host-encoded group II intron-like RTs that have acquired other functions often have substitutions for the conserved A residue in the YADD motif at the RT active site. This suggests that the active site can be modified by evolutionary pressures to suit diverse activities. This tolerance was exploited to mutate the highly conserved YADD motif to study structure-function relationships between conserved features of previously uncharacterized classes of RTs. Current laboratory data suggests that the YXDD motif can be modified to modulate different activities, although other crucial complementary or compensatory mutations are likely necessary. Through this work I obtained data that suggests group II intron RTs evolved from ancestral RdRPs, that their constricted active site might serve to increase fidelity, and that their conserved active site motif can be modified to modulate biochemical activities.