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 initiat ion using psoralen cross-linking. DNA templates containing cross-links at either -14/-13, -2/-1, or -4/-3 were constructed. These cross-link s are within the DNA-contacting region in the initiation complex. A cr oss-link at -2/-1 did not affect T7 RNA polymerase binding affinity, w hereas 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 bindin g nor the first RNA phosphodiester bond but, greatly inhibited further RNA chain extension. Circular dichroism spectroscopy revealed that DN A melting in the -4/-3 cross-link was greatly inhibited, indicating th at inhibition of RNA chain extension was a melting defect. Transcripti on shutoff on the -14/-13 cross-link may be due to inhibition of confo rmational changes in the polymerase-DNA complex. Because the -2/-1 cro ss-link is immediately upstream of the start site (+1), open complex f ormation may have been completely inhibited by this cross-link, accoun ting for the shutoff of transcription. Thus, depending on their locati on, psoralen cross-links affected different steps in the initiation pr ocess. We propose that promoter melting is progressive and that meltin g of one or two bp upstream of the +1 site is sufficient for formation of the first phosphodiester bond while further RNA chain extension wi thin the promoter depends on greater upstream melting of the promoter, which may be required for stabilization of the initiation complex.