Residues in the RNP1-like sequence motif of Rho protein are involved in RNA-binding affinity and discrimination

Area of Science:

• Molecular Biology
• Microbial Genetics

Background:

• Transcription termination in Escherichia coli is regulated by the Rho factor, a homohexameric protein.
• Rho factor interacts with nascent RNA to terminate transcription.
• Specific residues within Rho's RNA-binding domain are crucial for its function.

Purpose of the Study:

• To investigate the roles of Asp60 and Phe62 in the RNA-binding domain of Rho factor.
• To elucidate the contribution of these residues to Rho's RNA binding affinity and transcription termination activity.

Main Methods:

• Biochemical analysis of purified mutant Rho proteins (F62S Rho and D60G Rho).
• Assessment of RNA binding affinity using lambda cro RNA.
• In vitro transcription termination assays, with and without the NusG cofactor.

Main Results:

• Phe62 is essential for stabilizing Rho-RNA interactions via hydrophobic forces, as F62S Rho showed reduced RNA affinity and termination efficiency.
• Asp60 contributes to discriminating against non-productive RNA binding through electrostatic repulsion, as D60G Rho exhibited high affinity but poor specificity.
• Mutant Rho proteins displayed distinct defects in termination, with F62S Rho showing partial activity with NusG, while D60G Rho's efficiency was not improved by NusG.

Conclusions:

• Phe62 plays a critical role in Rho factor's ability to bind RNA stably.
• Asp60 is important for Rho's specificity in RNA binding, facilitating the dissociation of non-productive complexes.
• These findings clarify the distinct functional contributions of specific amino acid residues in Rho-mediated transcription termination.