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Characterization of a new rho mutation that relieves polarity of Mu insertions
1Department of Microbiology, University of Maryland at College Park 20742, USA.
Abstract:
We report the identification and characterization of a new rho mutation, rho614, that relieves polarity of Mu insertions in Escherichia coli. The mutation was identified by its ability to suppress the polarity of the Mu-mediated phi(lamB'-'lacZ)hyb61-4 fusion that is located at codon four of the lamB signal sequence. The rho614 mutation alters residue 80 in the proposed RNA-binding domain of Rho and is recessive to wild-type rho. We suggest that in the presence of the rho614 allele transcripts initiated at the Mu promoter PcM fail to terminate at presumptive Rho-dependent termination sites, namely rut1 and rut2, and continue into the 3' 'lamB gene allowing a LamB+ phenotype. This contention is supported by deletion analysis of the region and the observation that insertional inactivation of genes that reduce transcription from PcM, clpP (ATP protease), himA (IHF-alpha), and himD (IHF-beta), block the LamB+ phenotype. rho614, rho4 and rho201 alleles suppress the polarity of a malK::Mu insertion on the downstream lamB gene. However, the polarity of the phi(lamB'-'lacZ)hyb61-4 insertion is only suppressed by the rho614 mutation. We propose that the rho614 mutation allows suppression of transcriptional polarity without interfering with translation initiation signals of the truncated 'lamB gene. In addition to identifying a new rho mutation and Rho-dependent terminator sequence, this system provides a means of studying Rho protein/terminator relationships through the identification of new classes of rho mutations.
Insights
A new rho mutation, rho614, in Escherichia coli suppresses transcriptional polarity caused by Mu insertions. This finding aids in understanding Rho protein function and Rho-dependent termination.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Transcriptional polarity is a phenomenon where the expression of downstream genes is reduced or abolished.
- Bacteriophage Mu insertions can cause polar effects on gene expression in Escherichia coli.
- The Rho protein is a key bacterial transcription termination factor.
Purpose of the Study:
- To identify and characterize a novel mutation in the Rho factor that affects transcriptional polarity.
- To investigate the mechanism by which this mutation suppresses polarity of Mu insertions.
- To explore the relationship between Rho protein, terminator sequences, and transcriptional polarity.
Main Methods:
- Isolation and characterization of a new rho mutation (rho614) in Escherichia coli.
- Construction and analysis of Mu-mediated gene fusions (phi(lamB'-'lacZ)hyb61-4 and malK::Mu).
- Deletion analysis and insertional inactivation of genes involved in transcription (clpP, himA, himD).
Main Results:
- The rho614 mutation suppresses the polarity of specific Mu insertions, including phi(lamB'-'lacZ)hyb61-4.
- The rho614 mutation alters residue 80 in the Rho protein's RNA-binding domain.
- Transcripts initiated at the Mu PcM promoter appear to bypass Rho-dependent termination sites (rut1, rut2) in the presence of rho614.
- Reduced transcription from PcM or inactivation of him genes blocks the polarity suppression phenotype.
Conclusions:
- The rho614 mutation provides a novel tool for studying Rho-dependent transcription termination.
- This mutation allows for the suppression of transcriptional polarity without disrupting translation initiation signals.
- The study identifies new Rho-dependent terminator sequences and offers insights into Rho protein-terminator interactions.