Characterization of the rplB gene from Streptomyces collinus and its protein product by mass spectrometry.
Ribosomal protein L2 is the largest protein components of 50S subunits. The protein is implicated in peptidyl transferase activity and binds to functionally important domains of 23S rRNA. The rplB gene, which codes for ribosomal protein L2 was cloned from Streptomyces collinus. The gene rplB was isolated from BamHI fragment (3.0 kb) of chromosomal DNA possessing two partial and four complete ORF's in the order from 5' to 3': rplC, rplD, rplW, rplB, rpsS, and rplV. The gene organization corresponds to the S10 operon. Gene rplB (834 bp) encodes a polypeptide chain of 278 amino acids. The molecular mass calculated from genomic structure is 30.5 kDa and pI 11.87. Protein L2 is rich in positively charged amino acids (Arg 36, Lys 20, and His 11). N-terminal domain possesses topology similar to the oligonucleotide/oligosaccharide binding OB folds. The availability of genome sequence makes it possible to identify L2 protein by mass spectrometry, moreover it facilitates the characterization of its potential posttranslational modifications. To confirm the protein sequence derived from the rplB gene the tryptic peptides of L2 were analyzed by mass spectrometric techniques. The obtained data matched exactly with the results of DNA sequencing.
Mikulík
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Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague 4, Vídenská 1083, 142 20, Czech Republic. mikulik@biomed.cas.cz
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Halada
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D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D015153:Blotting, Western; D002853:Chromatography, Liquid; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis; D017434:Protein Structure, Tertiary / Q000235:genetics; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics*; D012270:Ribosomes / Q000737:chemistry; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D012269:Ribosomal Proteins; C029466:ribosomal protein L2