The possibility of using the 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as the affinity modificator of bacteriophage T7 DNA-dependent RNA polymerase was demonstrated. The UTP derivative used was rather efficient substrate substituting UTP in the transcription reaction performed by the enzyme. The UV treatment of "stopped" reaction complex formed using three of four substrate ribonucleotides, allow to obtain the covalent binding between the enzyme and the reaction product of 9 nucleotides length. The isolation and the analysis of the obtained "nucleotide-peptide" showed that the sequence of modified peptide corresponded to the fragment Tyr802-Lys826, which belonged to the conservative motif C in the enzyme structure. His811 or Asp812 residues belonging to this sequence are most probable targets of the modification.
Download full-text PDF |
Source |
|---|
Publication Analysis
Top Keywords
Similar Publications
TMPyP binding evokes a complex, tunable nanomechanical response in DNA.
Nucleic Acids Res
August 2024
Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó Str. 37-47, H1094 Budapest, Hungary.
TMPyP is a porphyrin capable of DNA binding and used in photodynamic therapy and G-quadruplex stabilization. Despite its broad applications, TMPyP's effect on DNA nanomechanics is unknown. Here we investigated, by manipulating λ-phage DNA with optical tweezers combined with microfluidics in equilibrium and perturbation kinetic experiments, how TMPyP influences DNA nanomechanics across wide ranges of TMPyP concentration (5-5120 nM), mechanical force (0-100 pN), NaCl concentration (0.
View Article and Find Full Text PDFThe crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins.
Nucleic Acids Res
May 2024
Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.
Article Synopsis
- RexA and RexB are part of an exclusion system that stops certain bacteriophage mutants from infecting Escherichia coli cells infected with a specific lambda phage.
- Recent studies reveal that RexA can bind to DNA independently of RexB, leaning the lambda switch towards lytic activity, but the precise molecular interactions behind this are not well-defined due to limited structural data.
- The authors present a detailed crystal structure of the RexA dimer, showing its two-domain setup and similarities to another protein, suggesting potential conformational changes necessary for DNA binding, with further evidence from mutagenesis studies highlighting its role in phage exclusion and bistable switch modulation.
Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates.
Methods Enzymol
November 2023
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States; Howard Hughes Medical Institute, Yale University, New Haven, CT, United States. Electronic address:
RNA is playing an ever-growing role in molecular biology and biomedicine due to the many ways it influences gene expression and its increasing use in modern therapeutics. Hence, production of RNA molecules in large quantity and high purity has become essential for advancing basic scientific research and for developing next-generation therapeutics. T7 RNA polymerase (RNAP) is a DNA-dependent RNA polymerase of bacteriophage origin and it is the most widely-utilized tool enzyme for producing RNA.
View Article and Find Full Text PDFA real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes.
Biochem Biophys Rep
September 2023
Department of Chemistry, 402 Wartik Laboratories, The Pennsylvania State University, University Park, PA, 16802, USA.
Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time.
View Article and Find Full Text PDFNatural History of DNA-Dependent DNA Polymerases: Multiple Pathways to the Origins of DNA.
Viruses
March 2023
Network of Researchers on the Chemical Evolution of Life (NoRCEL), Leeds LS7 3RB, UK.
One of the major evolutionary transitions that led to DNA replacing RNA as the primary informational molecule in biological systems is still the subject of an intense debate in the scientific community. DNA polymerases are currently split into various families. Families A, B, and C are the most significant.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!