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Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate
1Department of Metabolic Regulation, Boston Biomedical Research Institute, MA 02114.
Abstract:
Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA. To define the polyadenylylation site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coli deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-containing primer. The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis. Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript. In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the rho-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-loop of the primary transcript. A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonucleases compete for the 3' ends of mRNA molecules.
Insights
Researchers identified specific polyadenylation sites on bacterial messenger RNA (mRNA). This study reveals how poly(A) polymerase and exonucleases interact at mRNA 3' ends in Escherichia coli.
Area of Science:
- Molecular Biology
- Bacterial Genetics
Background:
- Bacterial mRNA is known to possess polyadenylate tails at its 3' end.
- The precise molecular structure and polyadenylation sites of bacterial poly(A) RNA remain largely uncharacterized.
Purpose of the Study:
- To elucidate the specific polyadenylation sites on bacterial messenger RNA (mRNA).
- To investigate the molecular structure of polyadenylated bacterial RNA, focusing on Escherichia coli lipoprotein mRNA.
Main Methods:
- Utilized Escherichia coli strains deficient in exoribonucleases to enrich for poly(A) RNA.
- Synthesized complementary DNA (cDNA) to polyadenylylated lipoprotein mRNA using reverse transcriptase and an oligo(dT) primer.
- Amplified cDNA using polymerase chain reaction (PCR) and sequenced the resulting DNA fragments cloned into pUC18.
Main Results:
- Identified two distinct polyadenylation sites within the 3' untranslated region of the lpp mRNA.
- Observed polyadenylation at the putative transcription termination site in some clones.
- Found polyadenylation preceding the stem-loop structure of the rho-independent transcription terminator in other clones.
Conclusions:
- Proposed a model where poly(A) polymerase and exonucleases compete for the 3' ends of bacterial mRNA.
- The findings provide insights into the regulation of bacterial mRNA processing and stability.