Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. II. Stability and some properties of elongation complexes.
We constructed a 66 base-pair DNA template capable of supporting transcription by T7 RNA polymerase. This template had a psoralen cross-link downstream from a T7 promoter such that a 36 (+1) nucleotide transcript was synthesized at the time the T7 polymerase came to a stop at the cross-link. The stability of elongation complexes formed on this template, and the effect of different factors that are known to affect polymerase-DNA interactions was investigated by non-denaturing gel electrophoresis and gel filtration chromatography. We found that an elongation complex could lose its RNA component but the T7 polymerase still remained attached to the DNA template for extended periods of time (at least up to 18 h). This type of an elongation complex, bereft of its nascent RNA transcript, is called a quasi-elongation complex. DNase I footprinting within gel slices indicated that the polymerase molecules were arrested at the psoralen cross-link on the DNA template in the quasi-elongation complexes. The quasi-elongation complexes were found to be extremely stable in 0.5 M-NaCl and in 0.2 M-NaCl plus 60 mM-MgCl2, and could withstand temperatures up to 42 degrees C. The quasi-elongation complexes were destabilized by heparin and excess calf thymus DNA. Excess tRNA caused only a minimal degree of disruption. Non-promoter-containing plasmid DNAs did not have a destabilizing effect on the quasi-elongation complexes. Interestingly, it was observed that in a T7 ternary transcriptional complex arrested by a psoralen cross-link, the nascent RNA transcript could be stabilized from release by the presence in trans of a plasmid DNA bearing a T7 promoter and a T7 terminator. Such a stabilization against RNA release was not observed with plasmid DNAs containing either only a promoter or a terminator. The elongation complexes were stable during gel filtration through Sephacryl S-300 HR. However, it was found that 30% to 45% of the labeled RNA was retained during gel filtration as RNA that was apparently free from ternary complexes.
Sastry
S S
SS
Department of Chemistry, University of California, Berkeley.
Hearst
J E
JE
D001483:Base Sequence; D002850:Chromatography, Gel; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000737:chemistry; D014764:Viral Proteins
D004279:DNA, Viral; D046911:Macromolecular Substances; D014764:Viral Proteins; D015636:Magnesium Chloride; C016507:hydroxymethyltrioxsalen; D012965:Sodium Chloride; D006493:Heparin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D005990:Glycerol; D014307:Trioxsalen