Mutational analysis and secondary structure model of the RNP1-like sequence motif of transcription termination factor...

Area of Science:

• Molecular Biology
• Bacterial Genetics
• Protein-RNA Interactions

Background:

• Transcription termination factor Rho from Escherichia coli binds nascent RNA for function.
• Rho contains a conserved sequence similar to the RNP1 motif, common in RNA-binding proteins.

Purpose of the Study:

• To investigate the roles of specific amino acid residues (Ile49-Ser67) in Rho's conserved sequence segment.
• To determine the impact of mutations on Rho's termination function, RNA binding, and cellular viability.

Main Methods:

• Random-sequence cassette mutagenesis was used to introduce mutations into the rho gene.
• Mutant phenotypes were assessed by cell survival at 42°C, termination efficiency at lambdatR1, mutant protein expression levels, and RNA binding ability.

Main Results:

• Residues within the RNP1-like sequence DGFGFLR (60-66) were more critical for termination and RNA binding than residues 49-59.
• Asp60, Phe62, and Arg66 were identified as particularly sensitive residues.
• Mutant properties align with a secondary structure model showing the RNP1-like sequence on a beta-strand.

Conclusions:

• The DGFGFLR motif is essential for Rho-dependent transcription termination and RNA interaction.
• Specific residues, Asp60, Phe62, and Arg66, play critical roles in Rho's function.
• The findings support a structural model of Rho involving beta-sheet interactions with RNA.