In vitro transcription close to the melting point of DNA: analysis of Thermotoga maritima RNA polymerase-promoter complexes at 75 degrees C using chemical probes.
The interaction of DNA dependent RNA polymerase of the extreme thermophile bacteria Thermotoga maritima with a promoter bearing DNA fragment was investigated in the temperature range from 20 to 85 degrees C. We show that the T. maritima RNA polymerase recognizes and utilizes the Escherichia coli T7 A1 promoter with an efficiency similar to that of the E. coli polymerase. We have investigated the interaction of both polymerases with the same promoter over a wide range of temperatures using hydroxyl radical foot-printing and osmium tetroxide probing. This study revealed that the T. maritima polymerase goes through a series of isomerisation events very similar to the E. coli polymerase, i.e. the closed, intermediate and open complexes, but the transitions themselves occur at radically different temperatures. This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence.
Meier
T
T
Max Planck Institute for Biochemistry, Martinsried, Germany.
Schickor
P
P
Wedel
A
A
Cellai
L
L
Heumann
H
H
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D007536:Isomerism; D008274:Magnesium; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009942:Organometallic Compounds; D011401:Promoter Regions, Genetic / Q000235:genetics; D013936:Thymidine / Q000032:analysis; D014158:Transcription, Genetic
D004279:DNA, Viral; D015335:Molecular Probes; D009942:Organometallic Compounds; D015082:2,2'-Dipyridyl; C076491:osmium tetroxide-2,2'-bipyridine; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D013936:Thymidine