PubMed Health⌕ Search

Biomedical subjects

S A Datwyler

Publications and source records attributed to S A Datwyler.

5 recordsLinked to original sources
PubMed2001

Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins.

The structure of an intermediate in the initiation to elongation transition of Escherichia coli RNA polymerase has been visualized through region-specific DNA cleavage by the hydroxyl radical reagent FeBABE. FeBABE was tethered to specific sites of the final sigma(70) subunit and incorporated into two specialized paused elongation complexes that obligatorily retain the final sigma(70) initiation subunit and are targets for modification by lambdoid phage late gene antiterminators. The FeBABE cleavage pattern reveals structures similar to open complex, except for notable changes to region 3 of final sigma(70) that might reflect the presence of stably bound transcript. Binding of the antiterminator protein Q displaces the reactivity of FeBABE conjugated to region 4 of final sigma(70), suggesting that final sigma(70) subunit rearrangement is a step in conversion of RNAP to the antiterminating form.

Bacteriophage lambda↗
PubMed2000

Protein-protein interactions mapped by artificial proteases: where sigma factors bind to RNA polymerase.

Interactions between proteins are important to understand but difficult to study. Conjugating a protein to a small artificial protease endows it with the ability to cut other proteins where it binds to them. Analysing the sites cut on the target proteins leads to new understanding of the structure of each complex. The binding of sigma factors to a common region on RNA polymerase provides an example.

Bacterial Proteins↗
PubMed1999

Targeted protein footprinting: where different transcription factors bind to RNA polymerase.

Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting to make an extensive survey of proximity to the protein surface. This involves attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues adjacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), NusA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large RNAP subunits, beta and beta', outlining a common surface. GreA cuts a larger set of sites, whereas omega shows no overlap with the others, cutting only the beta' subunit at a unique location.

Bacterial Proteins↗
PubMed1999

Mapping protein-protein interactions with a library of tethered cutting reagents: the binding site of sigma 70 on Escherichia coli RNA polymerase.

Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma 70 protein were conjugated with the cutting reagent iron (S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2-iminothiolane (2IT) with low efficiency. The result is a library of sigma 70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which sigma 70 binds. Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (alpha 2 beta beta') of Escherichia coli RNA polymerase. The beta subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H. The cut sites within the beta' subunit were intensely clustered between residues 250-450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G. No cut sites on the alpha subunit were observed. These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys sigma 70 mutants [Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis.

Binding Sites↗