Mutant bacteriophage T7 RNA polymerases with altered termination properties

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Bacteriophage T7 RNA polymerase (RNAP) is a crucial enzyme for transcription.
• RNAP pausing and termination are essential regulatory mechanisms in gene expression.
• Understanding these processes is key to controlling gene transcription.

Purpose of the Study:

• To identify and characterize mutants of T7 RNAP with altered termination and pausing abilities.
• To investigate the role of specific DNA sequences in T7 RNAP transcription termination.
• To explore the relationship between transcription initiation and termination.

Main Methods:

• Isolation and characterization of T7 RNAP mutants.
• In vitro transcription assays using various DNA templates (e.g., T7 DNA, rrnB, PTH gene).
• Analysis of enzyme processivity and interaction with T7 lysozyme.

Main Results:

• Mutant T7 RNAPs failed to terminate at specific sites (rrnB T1, PTH, CJ) but terminated normally at others (T(phi), rrnB T2).
• Mutants showed altered processivity on linear and supercoiled DNA and enhanced termination on poly(U) tracts.
• Mutations affected a region implicated in RNA binding and promoter contacts, suggesting a link between initiation and termination.

Conclusions:

• Specific mutations in T7 RNAP disrupt termination and pausing at defined signals.
• Termination may involve a reversal of initiation events, particularly in certain contexts.
• T7 lysozyme's effect on termination is linked to RNAP sensitivity and initiation complex stability.