A Novel Complex: A Quantum Dot Conjugated to an Active T7RNA Polymerase

To perform single-molecule studies of the T7RNA polymerase, it is crucial to visualize an individual T7RNA polymerase, for example, through a fluorescent signal. We present a novel complex combining two different molecular functions, an active T7RNA polymerase and a highly luminescent nanoparticle, a quantum dot. The complex has the advantage of both constituents: the complex can traffic along DNA and simultaneously be visualized, both at the ensemble and at the single-molecule level. The labeling was mediated through anin vivobiotinylation of a His-tagged T7RNA polymerase and subsequent binding of a streptavidin-coated quantum dot. Our technique allows for easy purification of the quantum dot labeled T7RNA polymerases from the reactants. Also, the conjugation does not alter the functionality of the polymerase; it retains the ability to bind and transcribe.

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Recent progress in single-molecule studies of mRNA localization in vivo

RNA Biology ◽

10.1080/15476286.2018.1536592 ◽

2018 ◽

Vol 16 (9) ◽

pp. 1108-1118 ◽

Cited By ~ 7

Author(s):

Songhee H. Kim ◽

Melissa Vieira ◽

Jae Youn Shim ◽

Hongyoung Choi ◽

Hye Yoon Park

Keyword(s):

Single Molecule ◽

Recent Progress ◽

Mrna Localization ◽

Single Molecule Studies

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RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6729 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6729-6735 ◽

Cited By ~ 32

Author(s):

J Liu ◽

W Zhou ◽

P W Doetsch

Keyword(s):

Rna Polymerase ◽

Rna Polymerases ◽

Dna Damages ◽

Mutant Proteins ◽

Dna Lesion ◽

Transcriptional Mutagenesis ◽

Rapid Generation ◽

Transition Mutation

Dihydrouracil (DHU) is a major base damage product formed from cytosine following exposure of DNA to ionizing radiation under anoxic conditions. To gain insight into the DNA lesion structural requirements for RNA polymerase arrest or bypass at various DNA damages located on the transcribed strand during elongation, DHU was placed onto promoter-containing DNA templates 20 nucleotides downstream from the transcription start site. In vitro, single-round transcription experiments carried out with SP6 and T7 RNA polymerases revealed that following a brief pause at the DHU site, both enzymes efficiently bypass this lesion with subsequent rapid generation of full-length runoff transcripts. Direct sequence analysis of these transcripts indicated that both RNA polymerases insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product. Such bypass and insertion events at DHU sites (or other types of DNA damages), if they occur in vivo, have a number of important implications for both the repair of such lesions and the DNA damage-induced production of mutant proteins at the level of transcription (transcriptional mutagenesis).

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Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets

Nucleic Acids Research ◽

10.1093/nar/gkz611 ◽

2019 ◽

Vol 47 (17) ◽

pp. e101-e101 ◽

Cited By ~ 3

Author(s):

Boris Breiner ◽

Kerr Johnson ◽

Magdalena Stolarek ◽

Ana-Luisa Silva ◽

Aurel Negrea ◽

...

Keyword(s):

Dna Sequencing ◽

Single Molecule ◽

Dna Polymerases ◽

Single Molecule Detection ◽

Synthesis Methods ◽

Single Nucleotide ◽

New Approach ◽

Fluorescent Signal ◽

Single Molecule Level ◽

Current Sequence

AbstractA new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

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Single-Molecule Studies of RNA Polymerases

Chemical Reviews ◽

10.1021/cr400207r ◽

2013 ◽

Vol 113 (11) ◽

pp. 8377-8399 ◽

Cited By ~ 14

Author(s):

Jens Michaelis ◽

Barbara Treutlein

Keyword(s):

Single Molecule ◽

Rna Polymerases ◽

Single Molecule Studies

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Single Molecule Studies on Hcv RNA Polymerase Activity

Biophysical Journal ◽

10.1016/j.bpj.2009.12.416 ◽

2010 ◽

Vol 98 (3) ◽

pp. 73a-74a

Author(s):

Pierre Karam ◽

Colins Vasquez ◽

Wayne Mah ◽

Megan Powdrill ◽

Matthias Götte ◽

...

Keyword(s):

Rna Polymerase ◽

Single Molecule ◽

Polymerase Activity ◽

Hcv Rna ◽

Single Molecule Studies ◽

Rna Polymerase Activity

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Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507592112 ◽

2015 ◽

Vol 112 (32) ◽

pp. E4390-E4399 ◽

Cited By ~ 152

Author(s):

Mathew Stracy ◽

Christian Lesterlin ◽

Federico Garza de Leon ◽

Stephan Uphoff ◽

Pawel Zawadzki ◽

...

Keyword(s):

Rna Polymerase ◽

Single Molecule ◽

Spatial Organization ◽

Search Time ◽

Population Level ◽

Bacterial Cells ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Superresolution Microscopy

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

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Single-Molecule Studies of RNA Polymerase: One Singular Sensation, Every Little Step It Takes

Molecular Cell ◽

10.1016/j.molcel.2011.01.008 ◽

2011 ◽

Vol 41 (3) ◽

pp. 249-262 ◽

Cited By ~ 69

Author(s):

Matthew H. Larson ◽

Robert Landick ◽

Steven M. Block

Keyword(s):

Rna Polymerase ◽

Single Molecule ◽

Single Molecule Studies

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A dual role for H2A.Z.1 in modulating the dynamics of RNA Polymerase II initiation and elongation

10.1101/2020.06.22.165373 ◽

2020 ◽

Author(s):

Constantine Mylonas ◽

Alexander L. Auld ◽

Choongman Lee ◽

Ibrahim I. Cisse ◽

Laurie A. Boyer

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Mammalian Cells ◽

Embryonic Stem ◽

Super Resolution ◽

Fine Tuning ◽

Transcriptional Initiation ◽

Single Molecule Level

AbstractRNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals1–5. During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome6–8, yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches2,9 (NET-seq and ChIP-nexus) along with live cell super-resolution microscopy (tcPALM)10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits re-assembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.

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Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

10.1101/855940 ◽

2019 ◽

Cited By ~ 1

Author(s):

Punam Sonar ◽

Wiphu Youyen ◽

Augustine Cleetus ◽

Pattipong Wisanpitayakorn ◽

Iman S. Mousavi ◽

...

Keyword(s):

Single Molecule ◽

Motor Coordination ◽

Intraflagellar Transport ◽

Kinetic Properties ◽

C Elegans ◽

Slow Velocity ◽

Single Molecule Studies ◽

Fast Transport ◽

And Function

SummaryThe construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors ‘line up’ in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other’s way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

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Nuclease dead Cas9 is a programmable roadblock for DNA replication

10.1101/455543 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kelsey Whinn ◽

Gurleen Kaur ◽

Jacob S. Lewis ◽

Grant Schauer ◽

Stefan Müller ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Fork ◽

Molecular Machines ◽

Replication Forks ◽

Chromosomal Dna ◽

Single Molecule Level ◽

Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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