TFIIIB subunit locations on U6 gene promoter DNA mapped by site-specific protein-DNA photo-cross-linking

ABSTRACT The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIEα closely approaches promoter DNA at nucleotide −10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIEα is obtained when the DNA template is artificially premelted in the −11/−1 region, suggesting that TFIIEα facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

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Identification and Topological Arrangement ofDrosophila Proximal Sequence Element (PSE)-Binding Protein Subunits That Contact the PSEs of U1 and U6 Small Nuclear RNA Genes

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1570 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1570-1579 ◽

Cited By ~ 20

Author(s):

Yan Wang ◽

William E. Stumph

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Sea Urchins ◽

Binding Protein ◽

Rna Polymerase Iii ◽

Specific Protein ◽

Cross Linking ◽

Sequence Element ◽

Proximal Sequence Element ◽

U6 Gene

ABSTRACT Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position −40 relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity ofDrosophila snRNA genes is intrinsically encoded in the PSE sequence itself. We have investigated the differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs. By using a site specific protein-DNA photo-cross-linking assay, we identified three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa that are in close proximity to the DNA and two additional putative polypeptides of 230 and 52 kDa that may be integral to the complex. The 95-kDa subunit cross-linked at positions spanning the entire length of the PSE, but the 49- and 45-kDa subunits cross-linked only to the 3′ half of the PSE. The same polypeptides cross-linked to both the U1 and U6 PSE sequences. However, there were significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding was to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters.

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Site-specific Protein Cross-Linking with Genetically Incorporated 3,4-Dihydroxy-L-Phenylalanine

ChemBioChem ◽

10.1002/cbic.200990036 ◽

2009 ◽

Vol 10 (9) ◽

pp. 1426-1426

Author(s):

Aiko Umeda ◽

Gabrielle Nina Thibodeaux ◽

Jie Zhu ◽

YungAh Lee ◽

Zhiwen Jonathan Zhang

Keyword(s):

Specific Protein ◽

Cross Linking ◽

Site Specific ◽

Protein Cross Linking

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Large-cluster and combined fluorescent and gold immunoprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166920 ◽

1996 ◽

Vol 54 ◽

pp. 892-893

Author(s):

Richard D. Powell ◽

James F. Hainfeld ◽

Carol M. R. Halsey ◽

David L. Spector ◽

Shelley Kaurin ◽

...

Keyword(s):

Metal Cluster ◽

Sulfonyl Chloride ◽

Gel Filtration ◽

Cross Linking ◽

Site Specific ◽

Fab Fragments ◽

Cluster Label ◽

Covalently Linked ◽

Texas Red ◽

Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

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Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

10.26434/chemrxiv.7150097 ◽

2018 ◽

Author(s):

Daniel D. Brauer ◽

Emily C. Hartman ◽

Daniel L.V. Bader ◽

Zoe N. Merz ◽

Danielle Tullman-Ercek ◽

...

Keyword(s):

Small Molecules ◽

Protein Modification ◽

Positive Charge ◽

Specific Protein ◽

High Yield ◽

Site Specific ◽

Protein Cage ◽

Protein Carriers ◽

Site Selective ◽

Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

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Faculty Opinions recommendation of Site-specific protein modification with a dirhodium metallopeptide catalyst.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13361053.14731199 ◽

2011 ◽

Author(s):

Burckhard Seelig ◽

Misha Golynskiy

Keyword(s):

Protein Modification ◽

Specific Protein ◽

Site Specific

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