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Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA
W J Meijer1, A Serna-Rico, M Salas
1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Abstract:
An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed.
Insights
The bacteriophage phi29 protein p16.7 is essential for early phage DNA replication. This membrane protein plays a crucial role in the infection process, particularly during the initial stages of viral DNA synthesis.
Area of Science:
- Molecular Biology
- Virology
- Bacteriophage Research
Background:
- Bacteriophage phi29 possesses an early expressed operon containing open reading frame (ORF)16.7.
- The deduced protein sequence of ORF16.7 is conserved across phi29-related phages.
Purpose of the Study:
- To characterize the protein encoded by ORF16.7 (p16.7) and elucidate its role in bacteriophage phi29 DNA replication.
- To investigate the membrane localization and potential dimerization of p16.7.
Main Methods:
- Expression and purification of a variant p16.7 protein (p16.7A) with a histidine-tag.
- Biochemical characterization of purified p16.7A, including dimerization analysis.
- Construction and analysis of a phi29 mutant with a mutation in gene 16.7 to study in vivo function.
Main Results:
- ORF16.7 encodes a membrane protein, p16.7, which is abundantly and early expressed post-infection.
- A transmembrane-spanning domain at the N-terminus is critical for p16.7 membrane localization.
- Purified p16.7A forms dimers in solution.
- phi29 DNA replication was impaired in the absence of p16.7, particularly at early infection stages.
Conclusions:
- p16.7 is an early expressed membrane protein crucial for efficient bacteriophage phi29 DNA replication.
- The N-terminal membrane anchor and potential dimerization may be important for p16.7 function.
- p16.7 likely plays a significant role in the early stages of phi29 DNA replication in vivo.