Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli

J Biol Chem. 1993 Nov 5;268(31):23477-82.

Abstract

The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cold Temperature
  • DNA-Directed RNA Polymerases / metabolism*
  • Escherichia coli / genetics
  • In Vitro Techniques
  • Oligonucleotides / metabolism
  • Osmolar Concentration
  • Promoter Regions, Genetic*
  • RNA, Ribosomal / genetics*
  • Sigma Factor / metabolism
  • Templates, Genetic
  • Transcription, Genetic*

Substances

  • Oligonucleotides
  • RNA, Ribosomal
  • Sigma Factor
  • DNA-Directed RNA Polymerases