DNA rearrangements generating artificial promoters

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The plasmid pBRH4, a derivative of pBR322, has a partially deleted tet gene promoter.
• This plasmid contains a functional Pribnow box near the EcoRI site, distinct from the inactivated tet promoter.

Purpose of the Study:

• To investigate the promoter activity of a cryptic sequence in pBRH4.
• To understand the formation and function of compound promoters involving this cryptic sequence.

Main Methods:

• Analysis of pBRH4 plasmid sequence and promoter elements.
• Site-directed mutagenesis to alter the -35 region.
• DNA deletion analysis around the HindIII site.
• Assessment of tet gene activation.

Main Results:

• A cryptic Pribnow box near the EcoRI site in pBRH4 was identified and shown to be functional.
• Compound promoters were formed when sequences homologous to the -35 region were adjacent to the cryptic Pribnow box, activating the tet gene.
• Point mutations in the nonfunctional -35 region of pBRH4 also activated the cryptic Pribnow box.
• Deletions near the HindIII site generated compound promoters by repositioning functional sequences relative to the -35 region.

Conclusions:

• The cryptic sequence in pBRH4 acts as an effective Pribnow box.
• Compound promoter formation is a mechanism for gene activation in this plasmid system.
• Deletions and mutations can be used to engineer novel promoter activities.