Deletion analysis of the Escherichia coli lactose promoter P2

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 PI) and an overlapping upstream promoter (termed P2). The structure of the P2 and the effect of RNA polyme...

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Bibliographic Details
Published in:Nucleic acids research 1985-04, Vol.13 (7), p.2457-2468
Main Authors: Yu, Xian-Ming, Reznikoff, William S.
Format: Article
Language:English
Subjects:
Base Sequence
beta-Galactosidase - genetics
DNA-Directed RNA Polymerases - metabolism
Endonucleases - metabolism
Escherichia coli
Escherichia coli - genetics
Exodeoxyribonucleases - metabolism
Gene Expression Regulation
Lac Operon
Macromolecular Substances
Mutation
Single-Strand Specific DNA and RNA Endonucleases
Transcription, Genetic
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Summary: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 PI) 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 PI 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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/13.7.2457