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.

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 seZB 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.
Selenoprotein synthesis requires a special translational step in which a specific UGA codon directs the incorporation of selenocysteine (1). In Escherichia coli, we could identify four genes, termed selA, selB, selC and selD, whose products are required for selenocysteine biosynthesis and incorporation (2). The gene product of selC is a tRNA species (tRNA& that is aminoacylated with L-serine (3). The products of the selA and selD genes are required for the subsequent conversion of serine to selenocysteine, which occurs on the tRNA (4, 5). A functional selB gene product is not required up to this step. In contrast, mutations in selB cause an accumulation of preformed selenocysteyl-tRNA"CA in uiuo (4  Fig. 1) contains a 2.1kilobase chromosomal DNA insert that complements a selB deletion mutation (2). Its nucleotide sequence was determined and revealed an open reading frame coding for a protein of 614 amino acids with a calculated size of 68.8 kDa (6). The putative selB gene was cloned into the expression vector pT7-6 (8) to give plasmid pWL194 in which the selB gene is under the control of the phage T7 410 promoter (see "Experimental Procedures").
In an in uiuo [35S]methionine-labeling experiment, induction of the T7 promoter led to the specific synthesis of a protein of 66.5 kDa ( Fig. 2A), which is in agreement with the size for SELB predicted from the nucleotide sequence.
Purification of SELB Protein-Purification of SELB was achieved after overproducing the protein using the T? expression system described above. E. coli strain K38 carrying plasmids pWL194 and pGPl-2 was grown in a lo-liter fermenter that had been induced to give high level expression of the selB gene. Since no activity assay was available for SELB quantitation, enrichment was followed by mixing the extract of these cells with a small amount of extract from cells that had been induced in the presence of [35S]methionine (incorporation was approximately 6.5 X 10' cpm) and by determin-  (7). Cell lysates were separated in a 10% polyacrylamide gel in the presence of sodium dodecyl sulfate (15). and the gel was dried and autoradiographed. Lane I, strain K38 carrying plasmids pGPl-2 and pT7-6 (vector control). Lane 2, K38/ nGPl-2/nWL184. B. nurification of SELB as followed bv SDSpolyacryiamide (lO%)'gel electrophoresis. Gels were stained with Coomassie Brilliant Blue R-250. Lane 1, molecular mass standards (a-galactosidase, bovine serum albumin, ovalbumin, and aldolase) lane 2, crude extract after 30,000 x g centrifugation; lane 3, supernatant of 150,000 X g centrifugation; lane 4, sediment of 150,000 X g centrifugation; lane 5, 1 M NH,Cl wash of 150,000 X g sediment; lane 6, 45-63% ammonium sulfate fraction; lane 7, hydroxylapatite pool; lane 8, purified SELB.
ing the distribution of radioactivity during fractionation. After optimization of the enrichment procedure, the final purifications were performed in the absence of radioactivity; Fig. 2B, gives a typical course of enrichment.
Briefly, the purification procedure can be summarized as sedimentation of SELB by a 60-min centrifugation at 150,000 x g, solubilization of the protein from the sediment by washing with 1 M NH&l, fractionated ammonium sulfate precipitation between 45 and 63% saturation, and hydroxylapatite chromatography. The final step, precipitation during dialysis against low salt buffer, takes advantage of the fact that SELB protein aggregates at a low salt concentration, e.g. in the presence of 10 mM potassium phosphate.
In a typical experiment, 21 mg of purified SELB was obtained from 4,725 mg of protein in a 30,000 X g supernatant. An enrichment factor of approximately 20-fold could be calculated from experiments in which [35S]methionine-labeled extract was used; the final yield was 9%.
The A280/A260 absorbance ratio of purified SELB was 1.62, demonstrating that it is essentially free of nucleotides. Gel filtration through a TSK G3000SW column in the presence of 0.02% polyoxyethylene 9 lauryl ether revealed that SELB, in its native state, is present as a monomer (results not shown).

N-terminalAmino
Acid Sequence of SELB-The nucleotide sequence that directly precedes the selB gene includes an open reading frame with a coding capacity for a protein of 463 amino acids (data not shown). It codes for the SELA protein, which has a function in the conversion of serine to selenocysteine (4, 5). The first potential ATG codon of the selB open reading frame overlaps by 4 base pairs with the TGA stop codon of the selA gene (Fig. 1). To determine exactly which codon initiates selB mRNA translation, the N-terminal amino acids of the purified protein were sequenced. The sequence obtained from 13 cycles was Met-Ile-Ile-Ala-Thr-Ala-Gly-His-Val-Asp-His-Gly-Lys. It is identical with the sequence derived from the DNA sequence of the selB gene starting with the first ATG codon as indicated in Fig. 1. This proves that (i) the ATG codon of selB, which overlaps with the TGA termination codon of the selA gene, is indeed used for initiation of selB mRNA translation; (ii) the N-terminal methionine is deformylated but not processed, and (iii) no frameshifting event takes place at the selA/selB overlap.
Limited Trypsinolysis of SELB-By analyzing the derived amino acid sequence of SELB, a striking similarity in primary structure was found with EF-Tu (6). As an initial means of analyzing conformational similarities between SELB and EF-Tu, SELB was subjected to limited proteolysis by trypsin. This approach has been used successfully for EF-Tu and EF-1 to evaluate the conformational state of these proteins (14,(18)(19)(20)(21)(22). Proteolysis of EF-Tu and other elongation factors results in a typical degradation pattern whereby the predominant cleavage occurs in a flexible protein fold near the N terminus which is exposed at the surface of the protein (14, 18-23). The experiment depicted in Fig. 3 demonstrates that SELB is also highly susceptible to low trypsin concentrations, yielding first a 63-kDa cleavage product followed by a 46-kDa degradation product. To define the sites within SELB at which cleavage occurs, the tryptic fragments were purified by reversed-phase HPLC on an RPC4 column (Fig. 4), and the N-terminal amino acids were sequenced. For the 63-kDa protein the sequence Gly-Met-Thr-Ile-Asp-Leu-Gly-Tyr-Ala-Tyr-Trp-Pro-Gln-Pro-Asp was obtained, which results from a cleavage between Arg34 and Gly35 in SELB. This site is homologous to the cleavage site Arg57-Gly58 in E. coli EF-Tu (23) and to similar cleavage sites in other elongation factors (14) (Fig. 5). The N-terminal sequence of the 46-kDa tryptic fragment was Gly-Met-Asp-Ala-Leu-(?)-Glu-(?)-Leu-Leu, which is the result of a cleavage between Arg15* and Gly153 in The experiment was carried out as described under "Experimental Procedures." Lane I, 4 pg of purified SELB prior to the addition of trypsin; lanes 2-5, after the addition of trypsin; lane 2, 5 min; lane 3, 10 min; lane 4, 20 min; lane 5, 40-min treatment with trypsin; lane 6, 4 pg of SELB incubated for 40 min at 28 "C in the absence of trypsin. The apparent molecular mass of SELB is indicated as deduced from its mobility in SDS-polyacrylamide gels. The molecular mass. as calculated from the amino ,cid sequence, is about 2.5 kDa larger. SELB. This specific cleavage site is not found in EF-Tu; however, in the tertiary structure model of EF-Tu proposed by La Cour et al. (24) and Jurnak (22), the homologous amino acids are situated in a small a-helical region (helix E) extending from the surface of the protein. From the fact that in SELB this second cleavage occurs rapidly and specifically, it can be concluded that the amino acids in this position are also exposed on the surface of the protein. The 2 glycine residues neighboring Arg"' in SELB probably promote better accessibility for trypsin in comparison with EF-Tu. These results are not only in agreement with the finding that SELB and EF-Tu share extensive similarity at the primary structure level, but they also suggest strongly that both proteins share common features in tertiary structure. Immunological Detection of SELB in E. coli Wild-type Cells-Antibodies directed against SELB were raised in rabbits and used to detect SELB protein in E. coli cells of strain FM420 (17) which is wild-type for selB. SDS-lysates of cells grown to exponential phase under aerobic and anaerobic conditions were subjected to immunoblot analysis. An estimation of the amount of SELB in these extracts was achieved by running different amounts of purified SELB protein on the same gel (Fig. 6A). The experiment showed that (i) SELB protein produced under conditions of wild-type level gene c w-- expression has an identical electrophoretic migration behavior when compared with overproduced SELB protein; (ii) only a single gene product results from selB gene expression, which is consistent with the N-terminal sequence data; (iii) SELB synthesis is not subject to aerobic/anaerobic regulation since essentially equal amounts of SELB could be detected in aerobic and anaerobic cells; (iv) the total cell lysates of 1.2 X lOa cells contains nearly 16 ng of SELB as determined by densitometric evaluation of the autoradiograph (using the Pharmacia LKB UltroScan XL), which is equivalent to about 1100 molecules of SELB/cell under the particular growth conditions (25). Thus, the ratio of SELB molecules to ribosomes is approximately 1:18 and that to EF-Tu, 1:180. The purification procedure had indicated that a major part of overproduced SELB protein co-sedimented with the ribosomes upon ultracentrifugation. In an attempt to determine the subcellular localization of SELB in cells of wild-type E. coli, extracts (30,000 x g supernatants) from strain FM420 of a Novel Translation Factor were prepared and loaded on a 30% sucrose cushion. After centrifugation for 16 h at 100,000 X g, the ribosomal pellet was washed with buffer containing 1 M NH&l, and the ribosomes were removed from the wash fluid by a further centrifugation step. Fig. 6B shows the distribution of SELB in the different fractions as determined by immunoblotting with anti-SELB antiserum. Approximately 50% of SELB was associated with the ribosomes and could be detached completely from the particulate fraction by washing with high salt. Whether this association reflects an intrinsic affinity of SELB to ribosomes remains to be demonstrated in uitro; however, such an observation is supportive of other data that favor the hypothesis that SELB is directly involved in translation (6).

K Forchhammer, K P Rücknagel and A Böck
involved in selenoprotein synthesis.