Erich E. Blatter

Domain organization of RNA polymerase α subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding

Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.

Identification of amino acid-base contacts in the Myc-DNA complex by site-specific bromouracil mediated photocrosslinking.

Myc binds to a 6 bp 2‐fold symmetric DNA site: 5′‐C‐3A‐2C‐1G+1T+2G+3‐3′. Using site‐specific 5‐bromouracil mediated photocrosslinking, we show that His336 of Myc contacts, or is close to, the thymine 5‐methyl group at 2‐fold symmetry‐related positions ‐2 and +2 of the DNA site in the Myc‐DNA complex. Our results strongly suggest that homologous amino acids of Myc and Max make equivalent contacts in the respective protein‐DNA complexes.

Localization of Cysteine 302 at the Active Site of Aldehyde Dehydrogenase

The superreactive cysteine was first identified in human cytoplasmic aldehyde dehydrogenase El isozyme, before its primary structure was known, as a part of 35 residue tryptic peptide (Hempel, 1981; Hempel and Pietruszko, 1981; Hempel et al., 1982) by employing iodoacetamide. When the primary structures of the El and E2 isozymes were established (Hempel et al., 1984, 1985; Hsu et al., 1985), this cysteine was found to occupy position 302 in a 500 amino acid residue polypeptide chain. Iodoacetamide fulfilled all criteria for an aldehyde-competitive, active-site-directed reagent with the exception of total inactivation of the mitochondrial E2 isozyme. Since that time, other investigators have also attempted to identify active site residues. Coenzyme-based affinity reagents (von Bahr-Lindstrom et al., 1985) identified cysteines 369 and 302, Nethylmaleimide identified cysteine 49 and 162 (Tu and Weiner, 1988 a,b) and dimethylaminocinnamaldehyde identified serine 74 (Loomes et al., 1990). Our laboratory developed a substrate-based affinity reagent, bromo-acetophenone (MacKerell et al., 1986), which identified glutamate 268 (Abriola et al., 1987).

Nitrate Esters as Inhibitors and Substrates of Aldehyde Dehydrogenase

Nitrate esters are used clinically as the smooth muscle relaxants (Needleman, 1975) in the treatment of angina pectoris. Guanine cyclase-mediated relaxation of aortic strips is dependent on the conversion of nitrate esters to nitric oxide by tissue sulfhydryl groups (Needleman and Johnson, 1973).