General Protein–Protein Cross-Linking

Alegria-Schaffer, Alice

doi:10.1016/b978-0-12-420120-0.00006-2

Cited by 7 publications

(5 citation statements)

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“…These 80-nt sequences are comprised of two 20-mer poly-A regions that alternate with two unique 20-mer sequences. The 5'-amine modification on these anchor sequences is necessary for amine-to-amine cross-linking 17 with PLL mediated by BS3. The second DNA patterning step does not require a cross-linker.…”

Section: Discussionmentioning

confidence: 99%

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Ramirez

Wang

2016

JoVE

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Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Ramirez

Wang

2016

JoVE

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“…50 µg from the most concentrated fractions were collected and used to crosslink the TEC to Spt4/5 using a BS 3 crosslinker (description of the crosslinking procedure is outlined in the section “Elongation complex assembly for cryo electron microscopy analysis”). BS 3 -crosslinking can stabilize the association for transcription factors, without influencing the architecture of their complexes 85 . Elution fractions from a second size exclusion chromatography run corresponding to the polymerase peak were collected and used for vitrification.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

Tarau,

Grünberger,

Pilsl

et al. 2024

Preprint

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Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery ofPyrococcus furiosus(Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly,PfuSpt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activityin vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

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“…50 μg from the most concentrated fractions were collected and used to crosslink the TEC to Spt4/5 using a BS 3 crosslinker (description of the crosslinking procedure is outlined in the section ‘Elongation complex assembly for cryo-electron microscopy analysis’). BS 3 -crosslinking can stabilize the association for transcription factors, without influencing the architecture of their complexes ( 60 ). Elution fractions from a second size exclusion chromatography run corresponding to the RNAP peak were collected and used for vitrification ( Supplementary Figure S3 ).…”

Section: Methodsmentioning

confidence: 99%

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

Tarău,

Grünberger,

Pilsl

et al. 2024

Nucleic Acids Research

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Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

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General Protein–Protein Cross-Linking

Cited by 7 publications

References 0 publications

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

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