Biomedical subjects

Reid C Johnson

Publications and source records attributed to Reid C Johnson.

22 records · Page 2Linked to original sources

PubMed2002

Architecture of Fis-activated transcription complexes at the Escherichia coli rrnB P1 and rrnE P1 promoters.

The transcription factor Fis activates the Escherichia coli rRNA promoters rrnB P1 and rrnE P1 by binding to sites centered at -71 and -72, respectively, and interacting with the C-terminal domain of the alpha subunit of RNA polymerase (RNAP alphaCTD). To understand the mechanism of activation by Fis at these promoters, we used oriented alpha-heterodimeric RNAPs and heterodimers of Fis to determine whether one or both subunits of alpha and Fis participate in the alphaCTD-Fis interaction. Our results imply that only one alphaCTD in the alpha dimer and only one activation-proficient subunit in the Fis dimer are required for activation by Fis. A library of alanine substitutions in alpha was used to identify the alphaCTD determinants required for Fis-dependent transcription at rrnB P1 and rrnE P1. We propose that the transcriptional activation region of the promoter-proximal subunit of the Fis dimer interacts with a determinant that includes E273 of one alphaCTD to activate transcription. We further suggest that the Fis contact to alphaCTD results in alphaCTD interactions with DNA that differ somewhat from those that occur at UP elements in the absence of Fis. The accompanying paper shows that the 273 determinant on alphaCTD is also targeted by Fis at the proP P2 promoter where the activator binds overlapping the -35 hexamer. Thus, similar Fis-alphaCTD interactions are used for activation of transcription when the activator is bound at very different positions on the DNA.

Base Sequence↗

PubMed2002

The C-terminal domains of the RNA polymerase alpha subunits: contact site with Fis and localization during co-activation with CRP at the Escherichia coli proP P2 promoter.

Fis is a versatile transactivator that functions at many different promoters. Fis activates transcription at the RpoS-dependent proP P2 promoter when bound to a site that overlaps the minus sign35 hexamer by a mechanism that requires the C-terminal domain of the alpha subunit of RNA polymerase (alphaCTD). The region on Fis responsible for activating transcription through the alphaCTD has been localized to a short beta-turn near the DNA-binding determinant on one subunit of the Fis homodimer. We report here that Fis-dependent activation of proP P2 transcription requires two discrete regions on the alphaCTD. One region, consisting of residues 264-265 and 296-297, mediates DNA binding. A second patch, comprising amino acid residues 271-273, forms a ridge on the surface of the alphaCTD that we propose interacts with Fis. The accompanying paper shows that these same regions on alphaCTD are utilized for transcriptional activation at the rrnB and rrnE P1 promoters by Fis bound to a site upstream of the core promoter (centered at minus sign71/minus sign72). In addition to stimulation of proP P2 transcription by Fis, CRP co-activates this promoter when bound to a remote site upstream from the promoter (centered at -121.5). RNA polymerase preparations lacking one alphaCTD of the alpha dimer were employed to demonstrate that the beta'-associated alpha(II)CTD was utilized preferentially by Fis at proP P2 in the presence and absence of CRP. These experiments define the overall architecture of the proP P2 initiation complex where Fis and CRP each function through a different alphaCTD.

Binding Sites↗

PubMed2002

Testing water-mediated DNA recognition by the Hin recombinase.

The Hin recombinase specifically recognizes its DNA-binding site by means of both major and minor groove interactions. A previous X-ray structure, together with new structures of the Hin DNA-binding domain bound to a recombination half-site that were solved as part of the present study, have revealed that two ordered water molecules are present within the major groove interface. In this report, we test the importance of these waters directly by X-ray crystal structure analysis of complexes with four mutant DNA sequences. These structures, combined with their Hin-binding properties, provide strong support for the critical importance of one of the intermediate waters. A lesser but demonstrable role is ascribed to the second water molecule. The mutant structures also illustrate the prominent roles of thymine methyls both in stabilizing intermediate waters and in interfering with water or amino acid side chain interactions with DNA.

Amino Acid Sequence↗

PubMed2002

The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly.

HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

Animals↗