New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.
The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.
Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.
Nicola
N
Doniselli
Jason L. Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, CA, USA Biophysics Graduate Group, University of California, Berkeley, CA, USA Laboratorio de Moléculas Individuales, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Av Honorio Delgado 430, San Martin de Porras, Lima-31, Peru.
Piere
P
Rodriguez-Aliaga
Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.
Davide
D
Amidani
Biophysics Graduate Group, University of California, Berkeley, CA, USA Laboratorio de Moléculas Individuales, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Av Honorio Delgado 430, San Martin de Porras, Lima-31, Peru.
Jorge A
JA
Bardales
Jason L. Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, CA, USA Biophysics Graduate Group, University of California, Berkeley, CA, USA Departments of Physics, Chemistry, and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA, USA carlosb@berkeley.edu.
Carlos
C
Bustamante
Laboratorio de Moléculas Individuales, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Av Honorio Delgado 430, San Martin de Porras, Lima-31, Peru.
Daniel G
DG
Guerra
Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy claudio.rivetti@unipr.it.
Claudio
C
Rivetti
D010582:Bacteriophage lambda / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020459:Genes, rRNA; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012265:Ribonucleotides / Q000378:metabolism; D061785:Transcription Initiation, Genetic; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012265:Ribonucleotides; C090751:dksA protein, E coli; D006159:Guanosine Tetraphosphate; D012321:DNA-Directed RNA Polymerases