[Phage T7 RNA-polymerase: gene cloning and its structure]

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• T7 phage encodes a specific RNA polymerase essential for its replication.
• Cloning phage genes into expression vectors facilitates study and production of viral proteins.

Purpose of the Study:

• To clone and express the T7 phage gene 1.0, responsible for T7 RNA polymerase production, in E. coli.
• To analyze and refine the sequence of the T7 gene 1.0.
• To investigate the functional consequences of sequence variations in the cloned gene.

Main Methods:

• Gene cloning into a plasmid vector (pSK-T7-1.0a and pSK-T7-1.0b).
• Bacterial expression of T7 RNA polymerase in E. coli K12 HB101.
• Enzyme activity assays (RNA synthesis stimulation).
• Restriction mapping of plasmids.
• DNA sequencing to identify mutations.

Main Results:

• Plasmid pSK-T7-1.0a successfully expressed active T7 RNA polymerase, confirmed by rifampicin-resistant RNA synthesis on a T7 DNA template.
• Sequence analysis of gene 1.0 from pSK-T7-1.0a allowed for refinement of its published sequence.
• Plasmid pSK-T7-1.0b, despite a similar restriction map, failed to produce active T7 RNA polymerase.
• Sequencing of pSK-T7-1.0b revealed a single thymine deletion in gene 1.0, explaining the loss of function.

Conclusions:

• The cloned T7 phage gene 1.0 in plasmid pSK-T7-1.0a encodes a functional T7 RNA polymerase.
• A single nucleotide deletion in T7 gene 1.0 can abolish RNA polymerase activity.
• This study refines the T7 gene 1.0 sequence and demonstrates the utility of plasmid-based expression for functional analysis.