scholarly journals The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies

2012 ◽  
Vol 102 (3) ◽  
pp. 484a
Author(s):  
Matthew L. Kahlscheuer ◽  
Nils G. Walter
2016 ◽  
Author(s):  
Armando de la Torre ◽  
Yoori Kim ◽  
Andrew A. Leal ◽  
Ilya J. Finkelstein

AbstractSingle-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. DNA isolated from bacteriophage λ (λ-DNA) is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific DNA sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into λ-DNA with 90-100% yield. Furthermore, various DNA structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of Proliferating Cell Nuclear Antigen (PCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that PCNA can load on both 5’-ssDNA flaps and a 13xCAG triplet repeat. However, PCNA remains trapped on the 13xCAG structure, confirming a proposed mechanism for triplet repeat expansion. Although we focus on λ-DNA, this method is applicable to all long DNA substrates. We anticipate that this molecular toolbox will be broadly useful for both ensemble‐ and single-molecule studies that require site-specific modification of long DNA substrates.


2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.


2010 ◽  
Vol 63 (4) ◽  
pp. 624
Author(s):  
Michael J. Serpe ◽  
Jason R. Whitehead ◽  
Stephen L. Craig

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.


2011 ◽  
Vol 100 (3) ◽  
pp. 464a
Author(s):  
Promod R. Pratap ◽  
Gregor Heiss ◽  
Martin Sikor ◽  
Don C. Lamb ◽  
Max Burnett

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