The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA.
An in vitro transcription system has been set up for formate- and FhlA-dependent transcription activation at the -12/-24 promoter of the fdhF gene from Escherichia coli by sigma 54-RNA polymerase. It requires the presence of the upstream activation sequence on supercoiled DNA. Transcription is independent from the effector formate at nucleoside triphosphate concentrations of 400 microM and above and completely dependent on the presence of the effector when the concentration is lowered to 300 microM. Inclusion of nucleoside diphosphates in the system raises the nucleoside triphosphate level at which specific induction by formate can take place. The threshold level of FhlA relative to that of template DNA required for transcription activation in the absence of formate was lowered at a high nucleoside triphosphate concentration. On the other hand, transcription activation at the fdhF promoter lacking the upstream activation sequence requires an increased ratio of FhlA to promoter plus the presence of formate; high ATP concentrations cannot bypass the effect of formate. These results are interpreted in terms of a model which implies that FhlA must undergo a change in its oligomeric state for transcription activation and that this oligomerization is favored by high nucleoside triphosphate concentrations, by the effector formate, and by the target DNA. In the absence of the target DNA, FhlA can line up at unspecific DNA and activate transcription; in this case, however, presence of formate and a higher FhlA concentration are required to stabilize and increase the amount of active oligomer.
Hopper
S
S
Lehrstuhl für Mikrobiologie der Universität München, Germany.
Korsa
I
I
Böck
A
A
D001483:Base Sequence; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005560:Formate Dehydrogenases / Q000235:genetics; D005561:Formates / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D009711:Nucleotides / Q000737:chemistry / Q000502:physiology*; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic / Q000235:genetics; D051877:RNA Polymerase Sigma 54; D012333:RNA, Messenger / Q000032:analysis; D012808:Sigma Factor / Q000502:physiology; D013698:Templates, Genetic; D015534:Trans-Activators / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000502:physiology*
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005561:Formates; D009097:Multienzyme Complexes; D009711:Nucleotides; D012333:RNA, Messenger; D012808:Sigma Factor; D015534:Trans-Activators; C497751:rpoN protein, E coli; C030544:formic acid; C067189:fhlA protein, E coli; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54