The Escherichia coli lactose (lac) operon transcription control region includes at least two sequences which are recognized by RNA polymerase holoenzyme in vitro, the normal lac promoter (termed P1) and an overlapping upstream promoter (termed P2). The structure of the P2 and the effect of RNA polymerase interaction at P2 on the association of RNA polymerase with P1 was analyzed by the isolation and characterization of various mutations at P2. A set of deletions with varying lengths of DNA between the lac P2 -10 region and a "-35 region" contributed by the vector DNA were constructed. In vitro studies indicate that as the spacing between the -10 region and "-35 region" is increased from 16 to 22 base pairs (bp), the steady state occupancy as measured by exonuclease III protection experiments and the ability to initiate transcripts from P2 decrease. Studies were also conducted using a single base pair insertion and a two base pair deletion between the natural -35 and -10 regions of P2. The mutation which decreases the in vitro occupancy and transcription initiation potential of P2 does not significantly affect the steady state in vitro occupancy of P1 nor the in vivo expression of the lac operon. These results are not consistent with the model that RNA polymerase occupancy at P2 competes with the P1 expression and therefore that this competition plays a role in cAMP bound catabolite gene activator protein (CAP-cAMP) control of the lac operon.