Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance

Biochemistry. 1998 Dec 22;37(51):18074-80. doi: 10.1021/bi9813431.

Abstract

Escherichia coli promoters for transcription of ribosomal and tRNAs are greatly activated by an A+T-rich "UP" element upstream of the -35 region. These same promoters have also been found to otherwise deviate in several respects from the consensus promoter sequence. Here we present the results of a kinetic characterization of the interaction of Escherichia coli RNA polymerase with UP element-containing promoters which by virtue of consensus or near-consensus sequence features should be among the most optimal that can be encountered by Escherichia coli RNA polymerase. We show that for such promoters, (1) the second-order rate constant describing formation of the initial (closed) complex is close to that expected for a diffusion-limited process, (2) the extent of activation by the UP element is temperature-sensitive, (3) the UP element accelerates a process after DNA binding by RNA polymerase, and (4) the presence of the UP element delays promoter clearance upon addition of nucleoside triphosphates to preformed RNA polymerase-promoter complexes. Finally, we provide evidence in support of models which describe the DNA melting process accompanying open complex formation as initiating in the -10 promoter region and progressing in the downstream direction.

Publication types

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

MeSH terms

  • Base Sequence
  • Consensus Sequence
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed RNA Polymerases / genetics*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Kinetics
  • Macromolecular Substances
  • Molecular Sequence Data
  • Peptide Chain Initiation, Translational / genetics
  • Promoter Regions, Genetic*
  • RNA / biosynthesis
  • Regulatory Sequences, Nucleic Acid*
  • Spectrometry, Fluorescence
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • rRNA Operon / genetics

Substances

  • DNA-Binding Proteins
  • Macromolecular Substances
  • Transcription Factors
  • RNA
  • DNA-Directed RNA Polymerases