Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Phage SP6 RNA polymerase is a crucial tool in molecular biology.
• Understanding RNA polymerase function, including termination and processivity, is essential for gene expression control.
• Developing methods to isolate specific polymerase mutants aids in studying enzyme mechanisms.

Purpose of the Study:

• To develop a system for isolating SP6 RNA polymerase mutants with altered termination proficiency and/or elongation processivity.
• To characterize the functional effects of identified mutations on termination at different intrinsic terminators and on elongation processivity.

Main Methods:

• Construction of a two-plasmid Escherichia coli system for in vivo mutant selection.
• Random mutagenesis of the SP6 RNA polymerase gene.
• In vivo selection based on LacZ activity and chloramphenicol resistance.
• In vitro characterization of purified mutant polymerases, including termination efficiency assays and transcript analysis.

Main Results:

• Three SP6 RNA polymerase mutants (M15L, M15S, D117G) were isolated.
• All mutants exhibited enhanced termination efficiency at the SP6 terminator and the upstream rrnB t1 signal.
• Mutations reduced termination efficiency at the downstream rrnB t1 signal and decreased overall elongation processivity.

Conclusions:

• The identified mutations reduce SP6 RNA polymerase elongation processivity.
• These mutations differentially affect termination at distinct intrinsic terminators, suggesting varied mechanisms.
• The findings imply different interactions between phage RNA polymerase and distinct termination signals.