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Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae
J Lingner1, J Kellermann, W Keller
1Department of Cell Biology, University of Basel, Switzerland.
Abstract:
Poly(A) polymerase is essential for the maturation of messenger RNA, adding tracts of adenosine residues to the 3' end of precursor RNA generated by endonucleolytic cleavage. This mechanism of mRNA 3' processing seems to be similar in yeast and in higher eucaryotes, although there are differences in the recognition signals in the pre-mRNA. Here we describe the cloning of the gene for yeast poly(A) polymerase. The enzyme is encoded by a single and essential gene located near the centromere on the left arm of chromosome 11. Poly(A) polymerase purified from recombinant Escherichia coli has the same physical and biochemical properties as the yeast enzyme. The yeast poly(A) polymerase shares features of sequence with its mammalian homologue.
Insights
Researchers cloned the gene for yeast poly(A) polymerase, an enzyme crucial for messenger RNA maturation. This essential enzyme shares sequence similarities with its mammalian counterpart, aiding in understanding mRNA processing across eukaryotes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Poly(A) polymerase is vital for messenger RNA (mRNA) maturation in eukaryotes.
- It adds adenosine residues to the 3' end of precursor RNA, a process involving endonucleolytic cleavage.
- While mRNA 3' processing is conserved, recognition signals in pre-mRNA differ between yeast and higher eukaryotes.
Purpose of the Study:
- To clone the gene encoding yeast poly(A) polymerase.
- To characterize the cloned yeast poly(A) polymerase.
- To compare yeast poly(A) polymerase with its mammalian homologue.
Main Methods:
- Gene cloning techniques were employed to isolate the yeast poly(A) polymerase gene.
- Recombinant expression in Escherichia coli was used to produce the enzyme.
- Purified recombinant enzyme was analyzed for physical and biochemical properties.
Main Results:
- The gene for yeast poly(A) polymerase was successfully cloned.
- The gene is a single, essential gene located on chromosome 11 near the centromere.
- Recombinant yeast poly(A) polymerase exhibited identical physical and biochemical properties to the native enzyme.
- Sequence analysis revealed conserved features between yeast and mammalian poly(A) polymerases.
Conclusions:
- The cloning and characterization of the yeast poly(A) polymerase gene provide a key resource for studying mRNA processing.
- The findings highlight evolutionary conservation in poly(A) polymerase structure and function between yeast and mammals.
- This work facilitates comparative studies of mRNA 3' end formation in different eukaryotic organisms.