| Autor: |
Tiggemann, Markus, Jeske, Stefanie, Larsen, Michael, Meinhardt, Friedhelm |
| Zdroj: |
Yeast; June 2001, Vol. 18 Issue: 9 p815-825, 11p |
| Abstrakt: |
The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactiswas expressed in Bacillus megateriumas a fusion protein with a His(6X)‐tag at the C‐terminus for isolation by Ni‐affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5′‐triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5′‐diphosphate end of the mRNA. Our in vitrodata provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid‐specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements. Copyright © 2001 John Wiley & Sons, Ltd. |
| Databáze: |
Supplemental Index |
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