Upstream promoter sequences and αCTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex

We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP–promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP α-subunit carboxy-terminal domain and on the presence and length of the RNAP α-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP–promoter open complex at the λ P(R) promoter is not a general feature of RNAP–promoter open complexes

Opening and closing of the bacterial RNA polymerase clamp.

Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes