Browsing by Subject "reverse transcriptase"

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Characterizing retron Efe1 reverse transcriptase interactions with ncRNA
(2024-04) Arguello, Carlos; Finkelstein, Ilya J.
Retrons are diverse bacterial anti-phage defense systems. A retron operon consists of a reverse transcriptase, an accessory protein, and a structured non-coding RNA that serves as the primer and template for reverse transcription. Retrons are currently being developed into new gene editing tools in bacteria, plants, and mammalian cells. A new retron system discovered in the Finkelstein lab, Efe1, has shown higher gene editing rates in mammalian cells than the current standard in retron gene editing, Eco1. Discovering what makes Efe1 better than Eco1 can elucidate the molecular mechanisms behind retron functions. Here, I investigate Efe1 reverse transcriptase and use cryo-electron microscopy to reconstruct a 3.9 Å density map of its RT-msDNA complex. Efe1 complex is highly similar to Eco1 complex, except that it is a monomer and its msDNA has a more rigid DNA stem loop than Eco1. Efe1 reverse transcriptase solubility decreases drastically in the absence of its cognate ncRNA. Efe1 reverse transcriptase can also be solubilized by Eco1 ncRNA and produce Eco1 msDNA. Mutating catalytic residues in Efe1 reverse transcriptase ablates msDNA production and reduces solubility. These findings offer insight into retron reverse transcriptase interactions with ncRNA that dictate proper protein folding and provide some guidance for future attempts at purifying retron reverse transcriptase alone.
Selection of RNA aptamers that inhibit mutant 3 HIV-1 reverse transcriptase
(2010) Husain, Naeem; Andrew D. Ellington
Acquired immunodeficiency syndrome (AIDS) is one of the most widespread diseases on the planet, and the need for new forms of treatment has become paramount. Even though there are treatments against human immunodeficiency virus (HIV), the virus mutates readily and becomes resistant to these drugs. A mutation is a slight change in the virus that allows it to evade the action of the medication. Because resistance to anti-viral drugs is pervasive, it is imperative to find new ways to treat such mutant viruses. A promising field for the treatment of HIV is the application of aptamers. Aptamers are nucleic acids that form tertiary structures, which can bind to proteins tightly and selectively. If an aptamer bound and inhibited a key enzyme in the virus, it could help prevent the spread of HIV in the human body. An important target for therapeutics has been reverse transcriptase, which is vital for viral reproduction. Although aptamers that inhibit reverse transcriptase (RT) activity have already been isolated, these are against wild type HIV, or most common form of the virus. Thus, it is important to select for alternative aptamers that will inhibit other drug resistant forms of the virus. The Stanford University HIV drug resistant database lists twelve common drug resistant HIV variants with mutant forms of RT. The object of this study was to select a few aptamers that inhibited the activity of mutant clone 3 RT from the aforementioned database. Aptamers were found not only to prevent enzymatic action of RT, but also bound to other regions of the protein itself. This led to the testing of innovative techniques within the realm of aptamers by creating complexes that link two aptamers together, which can bind more tightly to the enzyme because there are more contact points between the aptamers and protein target. These findings also open the door to improving current aptamer microarray assay technology. Microarrays utilize aptamers that are placed on slides to detect the presence of a certain protein. The improvement comes from using aptamers, instead of antibodies (another molecule that binds to proteins) as the signal molecule for the test. Thus, aptamers can be used for both therapeutic and detection means against HIV.
Using fluorescence tagging of amino acid residues distant from the active site of HIV-1 reverse transcriptase (RT) to obtain a unique fluorescent signal for nucleotide binding during catalysis
(2014) Jacobs, David Michael; Johnson, Kenneth A.
MDCC fluorophore labelling of HIV-1 RT has been used to monitor catalytic activity in the active site of the enzyme. Two fluorescence signals are generated in such experiments; one that results from the DNA template binding to the enzyme, and one that occurs when the next correct nucleotide binds to the enzyme-DNA complex and anneals to the nascent DNA strand. The intensity of the fluorescence signal resulting from the binding of the DNA template usually washes out the signal accompanying nucleotide binding. We hypothesized that by attaching MDCC to a residue on the surface of HIV-1 RT farther from the active site that underwent a drastic conformational change during catalysis, we would be able to generate a signal for nucleotide binding that was comparable in fluorescent intensity to the signal associated with DNA template strand binding. Our results for one labelling position were consistent with this hypothesis. Furthermore, our data confirms work conducted in past studies that showed how conformational changes in HIV-1 RT were related to catalytic activity at the active site. A brief literature review discussing past research describing this relationship, along with its implications, is included.