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NADH and its oxidized form NAD super(+) have a central role in energy metabolism, and their concentrations are often considered to be among the most important readouts of metabolic state. Here, we present a detailed protocol to image and monitor NAD super(+)/NADH redox state in living cells and in vivo using a highly responsive, genetically encoded fluorescent sensor known as SoNar (sensor of NAD(H) redox). The chimeric SoNar protein was initially developed by inserting circularly permuted yellow fluorescent protein (cpYFP) into the NADH-binding domain of Rex protein from Thermus aquaticus (T-Rex). It functions by binding to either NAD super(+) or NADH, thus inducing protein conformational changes that affect its fluorescent properties. We first describe steps for how to establish SoNar-expressing cells, and then discuss how to use the system to quantify the intracellular redox state. This approach is sensitive, accurate, simple and able to report subtle perturbations of various pathways of energy metabolism in real time. We also detail the application of SoNar to high-throughput chemical screening of candidate compounds targeting cell metabolism in a microplate-reader-based assay, along with in vivo fluorescence imaging of tumor xenografts expressing SoNar in mice. Typically, the approximate time frame for fluorescence imaging of SoNar is 30 min for living cells and 60 min for living mice. For high-throughput chemical screening in a 384-well-plate assay, the whole procedure generally takes no longer than 60 min to assess the effects of 380 compounds on cell metabolism.

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation. Inside cells, molecules of double-stranded DNA encode the instructions needed to make proteins. To make a protein, the two strands of DNA that make up a gene are separated and one strand acts as a template to make molecules of messenger ribonucleic acid (or mRNA for short). This process is called transcription. The mRNA is then used as a template to assemble the protein. An enzyme called RNA polymerase carries out transcription and is found in all cells ranging from bacteria to humans and other animals. Bacteria have the simplest form of RNA polymerase and provide an excellent system to study how it controls transcription. It is made up of several proteins that work together to make RNA using DNA as a template. However, it requires the help of another protein called sigma factor to direct it to regions of DNA called promoters, which are just before the start of the gene. When RNA polymerase and the sigma factor interact the resulting group of proteins is known as the RNA polymerase ‘holoenzyme’. Transcription takes place in several stages. To start with, the RNA polymerase holoenzyme locates and binds to promoter DNA. Next, it separates the two strands of DNA and exposes a portion of the template strand. At this point, the DNA and the holoenzyme are said to be in an ‘open promoter complex’ and the section of promoter DNA that is within it is known as a ‘transcription bubble’. However, it is not clear how RNA polymerase holoenzyme interacts with DNA in the open promoter complex. Bae, Feklistov et al. have now used X-ray crystallography to reveal the three-dimensional structure of the open promoter complex with an entire transcription bubble from a bacterium called Thermus aquaticus. The experiments show that there are several important interactions between RNA polymerase holoenzyme and promoter DNA. In particular, the sigma factor inserts into a region of the DNA at the start of the transcription bubble. This rearranges the DNA in a manner that allows the DNA to be exposed and contact the main part of the RNA polymerase. If the holoenyzyme fails to contact the DNA in this way, the holoenzyme does not bind properly to the promoter and transcription does not start. These findings build on previous work to provide a detailed structural framework for understanding how the RNA polymerase holoenzyme and DNA interact to form the open promoter complex. Another study by Bae et al.—which involved some of the same researchers as this study—reveals how another protein called CarD also binds to DNA at the start of the transcription bubble to stabilize the open promoter complex.

Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2',4'-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2',4'-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2',4'-bridged thymidine operates. With a Ki value of 9.7 plus or minus 1.1 MM, the 2',4'-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2',4'-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2',4'-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2',4'-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine thatThermus aquaticusMutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

The DNA polymerase derived from Thermus aquaticus is the most widely utilized among various DNA polymerases, indicating its significant economic importance. Consequently, efforts to achieve a substantial yield of Taq DNA polymerase (Taq-pol) are ongoing. The expression of recombinant protein using T7-induced promoters presents challenges in cost-effectiveness, primarily due to the reliance on traditional induction method. Our study aims to enhance cost-efficiency, and scalability of our method for overproducing Taq-pol, particularly in comparison to traditional IPTG-induced techniques, which remain underreported in the current literature. To achieve those purposes, this work integrated the use of (1) a high copy number vector; (2) an optimized chemically defined medium; and (3) optimized fermentation conditions in a 5 L bioreactor. A total of 83.5 mg/L of pure Taq-pol was successfully synthesized in its active form, leading to a 9.7-fold enhancement in protein yield. This was achieved by incorporating glucose, glycerol, and lactose into a defined medium at concentrations of 0.1, 0.6, and 1%, respectively, under specific production conditions in a 5 L bioreactor: 300 rpm, 2 vvm, and 10% inoculant. The data collectively suggest that the strategy serves as a significant foundation for the future advancement of large-scale production of Taq-pol.

The crystal structure of the initiating form of Thermus aquaticus RNA polymerase, containing core RNA polymerase ( alpha sub(2) beta beta ' omega ) and the promoter specificity sigma subunit, has been determined at 4 angstrom resolution. Important structural features of the RNA polymerase and their roles in positioning sigma within the initiation complex are delineated, as well as the role played by sigma in modulating the opening of the RNA polymerase active-site channel. The two carboxyl-terminal domains of sigma are separated by 45 angstroms on the surface of the RNA polymerase, but are linked by an extended loop. The loop winds near the RNA polymerase active site, where it may play a role in initiating nucleotide substrate binding, and out through the RNA exit channel. The advancing RNA transcript must displace the loop, leading to abortive initiation and ultimately to sigma release.

Allele-specific PCR is an important diagnostic tool that identifies single-nucleotide variants by preferential amplification of a particular allele, using primers that are mismatched to all but one allele variant. We applied a fluorescent stopped-flow polymerase assay to measure extension rates from oligonucleotide hairpins to simulate primer-template pairs. Under PCR-applicable conditions, reaction rates were recorded in nucleotides per second per polymerase (nt/s/poly). The effects of temperature, potassium chloride, mismatch type, and position were studied with primarily a deletion mutant of ( ) DNA polymerase and 135 oligonucleotide sequences. Rates at 65 °C were between 205 ± 11 and 177 ± 8 nt/s/poly for matched templates and between 4.55 ± 0.21 and 0.008 ± 0.005 nt/s/poly for 3'-mismatched templates. Although extension rates progressively increased with mismatches further away from the 3' end, rates were still reduced by as much as 84% with a C · C mismatch 6 bases from the 3' end. The optimal extension temperature for matched sequences was 70 °C, shifting to 55-60 °C for 3' mismatches. KCl inhibited mismatch extension. The Michaelis constant ( ) was increased and the apparent unimolecular rate constant (k ) decreased for 3' mismatches relative to matched templates. Although primer extension of mismatches depends on mismatch type and position, variation also depends on local sequence, KCl concentration, and the type of polymerase. Introduction of 3' mismatches reduces the optimal temperature for extension, suggesting higher annealing temperatures for better allele discrimination. Quantitative descriptions of expected specificity in allele-specific PCR provide additional design direction and suggest when other methods (e.g., high-resolution melting analysis) may be a better choice.