Related Experiment Videos
Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE-dependent endonucleolytic
Abstract:
Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities. As the result of a post-transcriptional processing event, a papA gene-specific mRNA product (mRNA-A) accumulates in large excess relative to the primary mRNA-BA transcript. Our results show that the processed product, mRNA-A, is a translationally active molecule and that it is generated from the mRNA-BA precursor by an RNaseE-dependent mechanism. The processing did not occur under non-permissive conditions in an E. coli rne mutant strain with a temperature-sensitive RNaseE. The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA. A comparison of nucleotide sequences of mRNA-BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites. Mutations abolishing the activity of either of two other endoribonucleases, RNaseIII and RNaseP, did not affect the pap mRNA processing event. However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E. coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency. Our findings are consistent with the idea that ams is related/allelic to rne. Absence of the processing event in the RNaseE mutant (rne-3071) strain led to a four-fold stabilization of the mRNA-BA primary transcript. We conclude that the RNaseE-dependent processing event is the rate-limiting step in the decay of the papB-coding part of the primary transcript and in the production of the stable mRNA-A product.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
The study reveals that Escherichia coli RNaseE enzyme processes pap mRNA, creating a stable mRNA-A product. This RNaseE-dependent processing is crucial for pap mRNA decay and the accumulation of translationally active mRNA-A.
Area of Science:
- Microbiology
- Molecular Biology
- Gene Expression Regulation
Background:
- Differential gene expression in Escherichia coli, particularly for Pap pili, is linked to mRNA stability.
- A specific papA gene mRNA (mRNA-A) accumulates due to post-transcriptional processing of a primary transcript (mRNA-BA).
Purpose of the Study:
- To elucidate the mechanism and key enzymes involved in the processing of pap mRNA in Escherichia coli.
- To determine the role of RNaseE in the generation and stability of pap mRNA products.
Main Methods:
- Utilized an Escherichia coli rne mutant strain with temperature-sensitive RNaseE to study pap mRNA processing.
- Investigated the effect of mutations in RNaseIII, RNaseP, and the ams locus on pap mRNA processing.
- Compared nucleotide sequences around cleavage sites of pap mRNA with other RNaseE-processed RNAs.
Main Results:
- The processed pap mRNA (mRNA-A) is translationally active and generated from mRNA-BA via an RNaseE-dependent pathway.
- Pap mRNA processing was abolished in the RNaseE mutant, leading to a four-fold stabilization of the primary mRNA-BA transcript.
- The ams locus mutation phenocopied RNaseE deficiency, suggesting ams is related or allelic to rne.
Conclusions:
- RNaseE-dependent processing is the rate-limiting step for the decay of the papB-coding region and the production of stable mRNA-A.
- This processing event is critical for regulating the abundance of functional pap mRNA in Escherichia coli.