Purification and biochemical characterization of SELB, a translation factor involved in selenoprotein synthesis.
The product of the selB gene from Escherichia coli is required for co-translational insertion of selenocysteine into protein. To make the SELB protein accessible to biochemical analysis, the protein was purified from cells that overexpressed the selB gene from a phage T7 promoter plasmid. It was calculated that the overproduced SELB protein was purified 20-fold. The N-terminal amino acid sequence of the purified protein was determined, and it confirmed that the initiation codon of selB mRNA translation overlaps the stop codon of the preceding selA gene by 4 bases. Structural similarity between SELB and elongation factors was demonstrated by limited proteolysis of SELB by trypsin. The cleavage sites within SELB were identified by N-terminal sequencing of the two proteolytic products. The position in the SELB protein of the major cleavage site was homologous to a tryptic cleavage site which is characteristic for elongation factors. Immunological analysis showed that the levels of SELB are equivalent in aerobically and anaerobically grown cells; the amount of the protein was estimated to be approximately 1100 copies/E. coli cell. Upon fractionation of cell extracts, SELB was found to be partially associated with the ribosomes. The results therefore indicate that SELB is the first known elongation factor-like protein that has specificity for a particular charged tRNA.
Forchhammer
K
K
Lehrstuhl für Mikrobiologie der Universität München, Federal Republic of Germany.
Rücknagel
K P
KP
Böck
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics / Q000302:isolation & purification*; D010446:Peptide Fragments; D010957:Plasmids; D014176:Protein Biosynthesis; D011506:Proteins; D012333:RNA, Messenger / Q000235:genetics; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine; D051140:Selenoproteins; D012689:Sequence Homology, Nucleic Acid; D013604:T-Phages / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D011506:Proteins; D012333:RNA, Messenger; C061878:SelB protein, Bacteria; D051140:Selenoproteins; D017279:Selenocysteine; D014357:Trypsin; D010444:Peptide Elongation Factor Tu; D012643:Selenium; D003545:Cysteine