Iodine-125 Radioprobing of E. coli RNA Polymerase Transcription Elongation Complexes

Abstract

Publisher Summary This chapter discusses the Iodine-125 Radioprobing of E. coli RNA polymerase transcription Elongation Complexes. Radioprobing of the Escherichia coli ( E. coli) RNA polymerase (RNAP) elongation complex demonstrate that approximately 9 bp upstream from the active site of RNAP, the DNA/RNA hybrid undergoes a drastic conformational change, and the two strands become separated. This result is in agreement with the crystal structure of the Pol II elongation complex, where the heteroduplex is 9 bp long, and the crystal/crosslinking-based model for the bacterial elongation complex, where the length of the heteroduplex is assumed to be 8-9 bp. The breaks distribution presented for the EC9 and EC10 is similar to that observed by the same method for the EC7 in the case of T7 RNAP. This non-A-like distribution of the breaks reflects the local separation of the RNA and the DNA T-strand. Therefore, the chapter concludes that the DNA/RNA hybrid formed inside the E. coli RNAP is 1-2 bp longer than the hybrid inside the T7 RNAP. The advantage of radioprobing over other foot printing and crosslinking methods is the ability of the DNA/RNA breaking agent—Auger electron—to freely penetrate proteins and nucleic acids.