Biomedical subjects
Publications and source records attributed to Ann Hochschild.
The bacteriophage lambda genetic switch is still yielding surprises. A recent study reveals that a long-range interaction involving proteins bound 2.4 kilobases away from one another on the phage genome mediates negative autoregulation, solving a long-standing puzzle concerning the regulation of lysogeny.
In bacteria, promoter recognition depends on the RNA polymerase sigma subunit, which combines with the catalytically proficient RNA polymerase core to form the holoenzyme. The major class of bacterial promoters is defined by two conserved elements (the -10 and -35 elements, which are 10 and 35 nucleotides upstream of the initiation point, respectively) that are contacted by sigma in the holoenzyme. We show that recognition of promoters of this class depends on the "flexible flap" domain of the RNA polymerase beta subunit. The flap interacts with conserved region 4 of sigma and triggers a conformational change that moves region 4 into the correct position for interaction with the -35 element. Because the flexible flap is evolutionarily conserved, this domain may facilitate promoter recognition by specificity factors in eukaryotes as well.
The Q protein of bacteriophage lambda is a transcription antiterminator that modifies the elongation properties of E. coli RNA polymerase (RNAP). To do this, DNA-bound (lambda)Q must first engage a paused elongation complex. Here we show that this engagement of (lambda)Q with RNAP involves an interaction between (lambda)Q and sigma(70), demonstrating that sigma(70) can be a target of regulation during elongation. Furthermore, we provide evidence that this interaction between (lambda)Q and sigma(70) stabilizes a conformation of RNAP that requires the disengagement of a segment of sigma(70) from the core enzyme. Recent structure-based models posit that the transition from the initiation to the elongation phase of transcription involves the staged displacement of sigma(70) from the RNAP core. Our findings provide support for this proposal.
Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the sigma70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with sigma70. We show here that the N-terminal half of MotA (MotA(NTD)), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of sigma70 with comparable sigma38 residues abolishes the interaction with MotA(NTD) in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA(CTD)) binds DNA with a K(D(app)) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of sigma70 helps to substitute functionally for an interaction between sigma70 and a promoter -35 element.
An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of gamma-secretase are discussed.