Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
49
|
| pubmed:dateCreated |
1997-1-16
|
| pubmed:abstractText |
Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at -4 was also slower in the presence of GTP than in its absence. At 37 degrees C, base pair disruption occurred first at -1 followed by -6 and finally at -4. Open complex formation was temperature-sensitive. Temperature effects at -1, -6, and at -4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around -1 and -6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at -1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near -1 and -6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at -1 and at -6, which are followed by bidirectional propagation to -4.
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| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/2-Aminopurine,
http://linkedlifedata.com/resource/pubmed/chemical/DNA,
http://linkedlifedata.com/resource/pubmed/chemical/DNA-Directed RNA Polymerases,
http://linkedlifedata.com/resource/pubmed/chemical/Fluorescent Dyes,
http://linkedlifedata.com/resource/pubmed/chemical/Guanosine Triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/Poly G,
http://linkedlifedata.com/resource/pubmed/chemical/Viral Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/bacteriophage T7 RNA polymerase
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Dec
|
| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
10
|
| pubmed:volume |
35
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
15715-25
|
| pubmed:dateRevised |
2008-11-21
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| pubmed:meshHeading |
pubmed-meshheading:8961934-2-Aminopurine,
pubmed-meshheading:8961934-Bacteriophage T7,
pubmed-meshheading:8961934-Base Composition,
pubmed-meshheading:8961934-DNA,
pubmed-meshheading:8961934-DNA-Directed RNA Polymerases,
pubmed-meshheading:8961934-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:8961934-Fluorescent Dyes,
pubmed-meshheading:8961934-Guanosine Triphosphate,
pubmed-meshheading:8961934-Hydrogen Bonding,
pubmed-meshheading:8961934-Kinetics,
pubmed-meshheading:8961934-Molecular Structure,
pubmed-meshheading:8961934-Nucleic Acid Conformation,
pubmed-meshheading:8961934-Nucleic Acid Denaturation,
pubmed-meshheading:8961934-Poly G,
pubmed-meshheading:8961934-Promoter Regions, Genetic,
pubmed-meshheading:8961934-Spectrometry, Fluorescence,
pubmed-meshheading:8961934-Spectrophotometry,
pubmed-meshheading:8961934-Temperature,
pubmed-meshheading:8961934-Thermodynamics,
pubmed-meshheading:8961934-Transcription, Genetic,
pubmed-meshheading:8961934-Viral Proteins
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| pubmed:year |
1996
|
| pubmed:articleTitle |
A direct real-time spectroscopic investigation of the mechanism of open complex formation by T7 RNA polymerase.
|
| pubmed:affiliation |
Laboratory of Molecular Genetics, Rockefeller University, New York, New York 10021, USA. sastrys@rockvax.rockefeller.edu
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| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
|