Interactions with Promoters

The overall process of transcription initiation and elongation is summarized in Figure 26.6. The first step in transcription is binding of RNA polymerase to DNA, followed by migration to an initiation DNA site called the promoter.

1. RNA polymerase finds promoters by a search process (Figure 26.6, step 1), in which the holoenzyme binds nonspecifically to DNA, with low affinity, and then slides along the DNA, without dissociation from it, until it reaches a promoter sequence, to which it binds with much higher affinity. factor is essential for this search, because the core enzyme does not bind to promoters more tightly than to nonpromoter sites. Binding to DNA and then moving along it reduce the complexity of the search for the promoter from three dimensions to one, just as finding a house becomes simpler once you find the street upon which that house is located.

2. The initial encounter between RNA polymerase holoenzyme and a promoter generates a closed-promoter complex. Whereas DNA strands unwind later in transcription, no unwinding is detectable in a closed-promoter complex. This complex forms with a Ka between 10⁶ and 10⁹ M^-1 at 0.1 M NaCl. Binding is primarily electrostatic, for Ka depends on ionic strength. The complex is relatively labile, dissociating with a half-life of about 10 seconds.

Footprinting studies (techniques that determine the region of DNA covered by a protein) show that polymerase contacts DNA from about nucleotide -55 to -5, where +1 represents the first DNA nucleotide to be transcribed.

3. RNA polymerase unwinds several base pairs of DNA, from about -10 to -1, giving an open-promoter complex, so-called because it binds DNA whose strands are open, or unwound. This highly temperature-dependent reaction occurs with half-times of about 15 seconds to 20 minutes, depending upon the structure of the promoter. The open-promoter complex is extremely stable; it is not easily disrupted by high ionic strength, and it forms with a Ka as high as 10¹⁴ M^-1. A Mg²⁺-dependent isomerization next occurs, giving a modified form of the open- promoter complex with the unwound DNA region extending from -12 to +2. Analysis indicates that DNA bending in the promoter region accompanies the transition from a closed-promoter to an open-promoter complex (Figure 26.8).