Overexpression of the methanococcal ribosomal protein L12 in Escherichia coli and its incorporation into halobacterial 50 S subunits yielding active ribosomes.
The gene for the ribosomal L12 protein from the archaebacterium Methanococcus vannielii was cloned into the expression vector pKK223-3. The protein was overexpressed and remained stable in Escherichia coli XL1 cells. Purification yielded a protein with the same amino acid composition and sequence as in Methanococcus but it was acetylated at the N terminus as in the case with the homologous protein of E. coli. The in vivo incorporation of the overexpressed protein into the E. coli ribosomes was not observed. The overexpressed M. vannielii protein MvaL12e was incorporated into halobacterial ribosomes, thereby displacing the corresponding halobacterial L12 protein. Intact 70 S ribosomes were reconstituted from halobacterial 50 S subunits carrying the MvaL12e protein. These ribosomes were as active as native halobacterial ribosomes in a poly(U) assay. On the other hand, our attempts to incorporate L12 proteins from Bacillus stearothermophilus and E. coli into halobacterial ribosomes were not successful. These results support the conclusion which is based on primary sequence and predicted secondary structure comparisons that there exist two distinct L12 protein families, namely the eubacterial L12 protein family and the eukaryotic/archaebacterial L12 protein family.
Köpke
A K
AK
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, West Germany.
Paulke
C
C
Gewitz
H S
HS
D000596:Amino Acids / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D019605:Euryarchaeota / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006217:Halobacterium / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure
D000596:Amino Acids; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12