In bacteria, RNA polymerase (RNAP) initiates transcription
by synthesizing short transcripts that are
either released or extended to allow RNAP to escape
from the promoter. The mechanism of initial transcription
is unclear due to the presence of transient
intermediates and molecular heterogeneity. Here,
we studied initial transcription on a lac promoter
using single-molecule fluorescence observations
of DNA scrunching on immobilized transcription
complexes. Our work revealed a long pause (‘‘initiation
pause,’’ �20 s) after synthesis of a 6-mer RNA;
such pauses can serve as regulatory checkpoints.
Region sigma 3.2, which contains a loop blocking
the RNA exit channel, was a major pausing determinant.
We also obtained evidence for RNA backtracking
during abortive initial transcription and for
additional pausing prior to escape. We summarized
our work in a model for initial transcription, in which
pausing is controlled by a complex set of determinants
that modulate the transition from a 6- to a
7-nt RNA