The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation.
The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.
Nickels
Bryce E
BE
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Garrity
Sean J
SJ
Mekler
Vladimir
V
Minakhin
Leonid
L
Severinov
Konstantin
K
Ebright
Richard H
RH
Hochschild
Ann
A
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D020035:Holoenzymes; D046912:Multiprotein Complexes; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit