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mRNA stabilizing signals encoded in the genome of the bacteriophage phi x174
M N Hayashi1, R Yaghmai, M McConnell
1Department of Biology, University of California, San Diego, La Jolla 92093.
Abstract:
In Escherichia coli cells infected with bacteriophage phi x174, mRNAs initiated by promoters PB and PD terminate after genes J, F, G, or H (TJ, TF, TG, or TH). These RNAs are relatively stable and contain mRNA-stabilizing signals at their 3' ends. These signals were cloned after gene D of phi x174 in an expression vector plasmid. The cloned signals stabilize mRNA of the upstream gene D and the stabilized mRNA is translationally functional. When these signals are inserted in reverse, no stabilizing effect on mRNA is observed indicating that the correct sequences at the 3' ends of transcripts determine their stability. When a stabilizing signal (+) and a mutated stabilizing signal (-) which has reduced stabilizing activity are tandemly inserted after gene D, two sets of 3' termini of the transcript are observed indicating that both signals also function as terminators. The amount of gpD synthesized from these constructs varies depending upon the relative positions of the (+) or (-) signals after gene D. The stabilizing function seems to act by preventing mRNA degradation from the 3' to 5' direction. Several common features of these stabilizers are described.
Insights
Bacteriophage phi x174 mRNA 3' end signals stabilize gene D mRNA in Escherichia coli, enhancing protein production. These sequences also act as terminators, with their orientation and position affecting expression levels.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phi x174 infection in Escherichia coli leads to mRNA termination at specific sites (TJ, TF, TG, TH).
- These terminated RNAs exhibit stability due to 3' end signals.
Purpose of the Study:
- To investigate the function of 3' end mRNA-stabilizing signals from bacteriophage phi x174.
- To determine if these signals can enhance gene expression when cloned into an expression vector.
Main Methods:
- Cloning of phi x174 3' end signals downstream of gene D in an expression plasmid.
- Analysis of mRNA stability and translational activity of the modified constructs.
- Insertion of wild-type and mutated stabilizing signals in tandem to assess terminator function.
Main Results:
- Cloned 3' end signals stabilized upstream gene D mRNA, maintaining translational functionality.
- Reversed signal insertion abolished the stabilizing effect, confirming sequence-specificity.
- Both stabilizing and mutated signals functioned as terminators, influencing transcript 3' end formation.
- gpD synthesis varied based on the position and type of inserted signals.
Conclusions:
- 3' end sequences of bacteriophage phi x174 transcripts possess mRNA-stabilizing and terminator functions.
- Signal orientation is critical for stabilization, suggesting a 3' to 5' degradation prevention mechanism.
- These signals can be exploited to modulate gene expression in prokaryotic systems.