Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis.
We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, approximately 70%-90%, of early elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region increases sigma70 retention in early elongation complexes. The results establish that a significant fraction, approximately 50%-60%, of mature elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region does not appreciably affect sigma70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma70, the half-life of sigma70 retention is long relative to the time-scale of elongation, suggesting that some complexes may retain sigma70 throughout elongation.
Kapanidis
Achillefs N
AN
Department of Chemistry and Biochemistry, and California Nanosystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, USA. a.kapanidis1@physics.ox.ac.uk
Margeat
Emmanuel
E
Laurence
Ted A
TA
Doose
Sören
S
Ho
Sam On
SO
Mukhopadhyay
Jayanta
J
Kortkhonjia
Ekaterine
E
Mekler
Vladimir
V
Ebright
Richard H
RH
Weiss
Shimon
S
D001681:Biological Assay / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D012808:Sigma Factor; D012319:RNA Polymerase II; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases