Probing the importance of selected phylum-specific amino acids in sigma A of Bacteroides fragilis, a primary sigma factor naturally devoid of an N-terminal acidic region 1.1.
The sigmaA factor of Bacteroides fragilis is the prototype of a novel subgroup of primary sigma factors that are essential for growth and ensure the initiation of transcription of the housekeeping genes. This subgroup is confined to the phyla Bacteroidetes and Chlorobi. Its members carry a specific amino acid signature and are notably characterized by a short, basic N-terminal segment instead of the typical acidic region 1.1. Using in vitro mutagenesis, we investigated the importance of this basic segment and of several residues of the signature for the function of sigmaA. We have shown that the conserved residues Phe-61 and Lys-265, located in the core binding and DNA binding subregions 2.1 and 4.2, respectively, are critical for full function of the B. fragilis holoenzyme. With respect to the unusual subregion composition of sigmaA, we have shown that truncation of the basic N-terminal segment, or reversion of its charge, strongly affects the overall transcriptional activity of B. fragilis RNA polymerase in vitro. Our results indicate that the presence of the intact basic segment is required for the formation of RNA polymerase (RNAP)-promoter open complexes, the correct architecture of the transcription bubble, and efficient promoter clearance.
Vingadassalom
Didier
D
Université Paris 6, Paris F-75006, France.
Kolb
Annie
A
Mayer
Claudine
C
Collatz
Ekkehard
E
Podglajen
Isabelle
I
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001441:Bacteroides fragilis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D041963:Bacteroidetes / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D019414:Chlorobi / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology
D000596:Amino Acids; D010446:Peptide Fragments; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases