Cro regulatory protein specified by bacteriophage lambda. Structure, DNA-binding, and repression of RNA synthesis

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophage lambda's Cro protein regulates early gene expression and is crucial for lytic growth.
• Understanding Cro protein's function is key to deciphering viral replication strategies.

Purpose of the Study:

• To purify and characterize the Cro protein from bacteriophage lambda.
• To analyze its DNA-binding properties and repressor activity.
• To compare Cro protein's function with the lambda cI repressor.

Main Methods:

• Purification of Cro protein to homogeneity.
• Analysis of protein structure and DNA-binding affinity using purified components.
• In vitro transcription assays to assess repressor activity.
• Site-directed mutagenesis to confirm gene product identity.

Main Results:

• Purified Cro protein exists as a dimer with subunits of 8600 molecular weight.
• Cro protein binds specifically to lambda-DNA operator sites (oL and oR) with high affinity (Kd ~10^-10 to 10^-11 M).
• Cro protein effectively represses transcription of N and cro genes, demonstrating autorepression.
• A missense mutation in the cro gene resulted in a temperature- and salt-sensitive DNA-binding product.

Conclusions:

• Cro protein is an autorepressor that regulates its own synthesis and early viral gene expression.
• Cro acts as a "weak repressor," distinct from the "strong repressor" cI protein.
• Cro's function is specialized for gradual repression of early genes, promoting the late stage of lytic development.