Probing the mechanisms of T7 RNA polymerase transcription initiation using photochemical conjugation of psoralen to a promoter.
We have dissected the steps in T7 RNA polymerase transcription initiation using psoralen cross-linking. DNA templates containing cross-links at either -14/-13, -2/-1, or -4/-3 were constructed. These cross-links are within the DNA-contacting region in the initiation complex. A cross-link at -2/-1 did not affect T7 RNA polymerase binding affinity, whereas a cross-link at -14/-13 reduced binding affinity by less than 2-fold. Transcription initiation was completely blocked by cross-links at -14/-13 or at -2/-1. A cross-link at -4/-3 inhibited neither binding nor the first RNA phosphodiester bond but greatly inhibited further RNA chain extension. Circular dichroism spectroscopy revealed that DNA melting in the -4/-3 cross-link was greatly inhibited, indicating that inhibition of RNA chain extension was a melting defect. Transcription shutoff on the -14/-13 cross-link may be due to inhibition of conformational changes in the polymerase-DNA complex. Because the -2/-1 cross-link is immediately upstream of the start site (+1), open complex formation may have been completely inhibited by this cross-link, accounting for the shutoff of transcription. Thus, depending on their location, psoralen cross-links affected different steps in the initiation process. We propose that promoter melting is progressive and that melting of one or two bp upstream of the +1 site is sufficient for formation of the first phosphodiester bond while further RNA chain extension within the promoter depends on greater upstream melting of the promoter, which may be required for stabilization of the initiation complex.
Sastry
S S
SS
Laboratory of Molecular Genetics, Rockefeller University, New York, New York 10021, USA. sastrys@rockvax.rockefeller.edu
Ross
B M
BM
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D008730:Methoxsalen / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010777:Photochemistry; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011489:Protein Denaturation; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects* / Q000528:radiation effects; D014764:Viral Proteins
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008730:Methoxsalen