Primary structure of the archaebacterial Methanococcus vannielii ribosomal protein L12. Amino acid sequence determination, oligonucleotide hybridization, and sequencing of the gene.
The primary structure of ribosomal protein L12 from Methanococcus vannielii has been determined by direct amino acid sequence analysis with automated liquid phase Edman degradation of the entire protein and manual 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate sequencing of fragments obtained by enzymatic digestion and by partial acid hydrolysis. The knowledge of the amino acid sequences of these various fragments allowed the synthesis of two oligonucleotide probes complementary to the 5'- and the 3'-end of the gene, and they were used for hybridization with digested M. vannielii chromosomal DNA. Both oligonucleotide probes gave similar and clear hybridization signals. The plasmid pMvaX1 containing the entire gene of protein L12 was obtained. The nucleotide sequence complemented the partial amino acid sequence, and it is in full agreement with the protein sequence and the amino acid analysis. Comparison of secondary structural elements and hydrophobicity plots of the M. vannielii protein L12 with the known L12 sequences derived from other archaebacterial and eukaryotic sources show strong homologies among these sequences. They contain an exceptional highly conserved hydrophilic sequence area in the C-terminal part of the proteins. In comparison with eubacterial L12 proteins, the conservation is reduced to single amino acid residues. However, the eubacterial L12 proteins have hydrophilic regions similar to those of L12 from M. vannielii. These regions are predicted to be located at the surface of the proteins, as has been proven to be the case in crystallized Escherichia coli L12 protein. It is possible that the strongly conserved hydrophilic sequence regions form part of the factor-binding domain.
Strobel
O
O
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, West Germany.
Köpke
A K
AK
Kamp
R M
RM
Böck
A
A
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D019605:Euryarchaeota / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000235:genetics*; D013045:Species Specificity
D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D004262:DNA Restriction Enzymes