title: Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage.
abstract: The alpha subunit of Escherichia coli RNA polymerase plays essential roles in protein-protein contacts, not only for RNA polymerase assembly, but also for transcription activation by class I factors. To reveal the structure-function relationship of the alpha subunit, we attempted to elucidate the organization of the structural domains by analysis of the pattern of limited proteolysis with two endoproteases, V8 protease and trypsin. The results indicate that one region, Arg235 to Glu244, is highly accessible to endoproteases. We propose that the alpha subunit consists of two major structural domains, the amino-terminal domain upstream from Arg235 and the carboxy-terminal domain downstream from Glu245, each being connected by an inter-domain linker formed by the spacer between these two amino acid residues. The structural organization is in good agreement with its functional map, i.e., the amino-terminal subunit assembly determinants and the carboxy-terminal transcription activation determinants, including the contact sites with class I transcription factors and DNA UP (enhancer) elements. The secondary proteolytic cleavage sites were also determined, in order to analyse intra-domain structures.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Negishi
authors.forename: N
authors.initials: N
authors.lastname: Fujita
authors.forename: A
authors.initials: A
authors.lastname: Ishihama
mesh_terms: D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D012321:DNA-Directed RNA Polymerases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin