Molecular Microbiology (1992) 6(23), 3461-3467
Structure of selenocysteine synthase from Escherichia coli and location of tRNA in the seryi-tRNA^^'^-enzyme complex Harald Engelhardt,^* Karl Forchhammer,^ Shirley Muller,^ Kenneth N. Goldie^ and August 'Max-Planck-lnstitut fur Biochemie, D-8033 Martinsried, Germany. ^Lehrstuhl fur f^ilirobiologie der Universitat, D-8000 Munchen 19, Maria-Ward-Strasse la, Germany. ^M.E. Muller-Institute for High-Resolution Electron hAicroscopy at fhe Biocenfre, University of Basel, Ch-4056 Basel, Switzerland. Summary Selenocysteine synthase of Escherichia coii catalyses the biosynthesis of selenocysteine in the form of the aminoacyMRNA complex, the reaction intermediate being aminoacrylyl-tRNA*^" covalently bound to the prosthetic group of the enzyme. Selenocysteine synthase and the specific aminoacrylyl-tRNA^^'' -enzyme complex as well as the isolated seryltRNA^^*^ were investigated in the electron microscope and analysed by means of image processing to a resolution of 2 nm in projection. The stoichiometric composition of the selenocysteine synthase molecule was elucidated by scanning transmission electron microscopic mass determination. The enzyme has a fivefold symmetric structure and consists of 10 monomers arranged in two rings. The tRNA is bound near the margin of the dimeric subunits. Principal component analysis of the tRNA-enzyme complexes revealed that the selenocysteine synthase appears to bind only one seryl-tRNA^*" per dimer, which is consistent with the result of biochemical binding studies.
{Leinfelder et ai, 1988). SeryMRNA^^^ is subsequently converted to selenocysteyl-tRNA^^'' by the novel enzyme selenocysteine synthase through pyridoxal phosphatedependent catalysis (Forchhammer and Bock, 1991). Selenocysteine synthase, the product of gene selA, specifically complexes charged and free tRNA^^'^ and thus functions in tRNA^*"' identification. When the enzyme binds seryl-tRNA^^*^, the substrate is converted to the stable aminoacrylyl-tRNA^^'' intermediate linked to pyridoxal phosphate. Addition of the reactive selenophosphate molecule {Ehrenreich et ai. 1992; Veres et ai, 1992), provided by the protein SELD, generates selenocysteyl-tRNA'^^'' which is released from the enzyme (Forchhammer and Bock, 1991). Selenocysteine synthase is one of the few enzymes that bind aminoacyl-tRNA as a substrate. The tRNA^^"" has a mass and dimensions large enough to be visualized in the electron microscope by appropriate preparation techniques. Usually nucleic acids are rendered visible by heavy-metal shadowing (Kay, 1976), dark-field electron microscopy or labelling by antibodies (Kastner ef ai, 1992). In this study we have successfully applied negative staining to investigate isolated tRNA and tRNA-protein complexes to a resolution of about 2 nm. The stability of the covalent aminoacrylyl-tRNA^^'^-selenocysteine synthase complex enabled us to localize the tRNA^®'' molecules on the enzyme. We also present the projected structure and the stoichiometric composition of the novel enzyme selenocysteine synthase.
Results and Discussion Structure and stoichiometry of the selenocysteine synthase
Introduction The 21st amino acid encoded by the DNA is selenocysleine, a functionally indispensable constituent of several enzymes in eukaryotes, eubacteria and archaebacteria (Bock etai, 1991), In Esc/7enc/i/aco//the biosynthesis of selenocysteine was elucidated recently; it proceeds in two steps. First, L-serine is esterified to selenocysteinespecitic tRNA''^'' by the cellular seryl-tRNA synthetase
Received 1 June, 1992; revised and accepted 22 August, 1992, 'For correspondence. Tel, (89) 85782650; Fax (89) 85782641,
The selenocysteine synthase molecules were imaged in the electron microscope using various heavy-metal salts as negative stains. While ail the stains gave sufficient contrast they had different effects on the orientation of the molecules on the specimen support. Uranyl acetate (pH 4.5) nearly exclusively yielded top-view orientations of the selenocysteine synthase (Fig, 1) while uranyl oxaiate (pH 6.8) resulted in 60-70% side-view orientation. The tRNA-enyzme complexes tended to edge-on orientation throughout with an extreme example for phosphotungstate (pH 6.8) where only 10% of the complexes
3462
H. EngelhardteXa\.
100
200 300 400 500 BOO 700 Mass IkDa)
Fig. 1. Electron microscopic imaging and mass determination ofthe selenocysteine synthase from E. coli. The scale bars represent 100 nm. A. Bright-field electron micrograph of negatively stained selenocysteine synthase showing top-view and side-on view (arrow) projections, B. STEM dark-field micrograph of unstained, freeze-dried moiecules recorded at 350 electrons nm"^. The inset shows the average of 230 molecules, exhibiting a distinct central channel, C. Histogram of mass values extracted from STEM images such as shown in (B), The average of 433±57 kDa is not corrected for beam-induced mass loss which is significant at a recording dose of 300 to 400 electrons/nm^.
appeared in the top-view orientation. In the following uranyt acetate and uranyl oxaiate preparations were analysed. The selenocysteine synthase of E coti is a fivefold symmetric molecule in projection (Figs 1 and 2). Five distinct bi-lobed, cloverleaf-like subunits are connected at thieir bases in a ring-like manner. The enzyme has a diameter of 19 nm and a central hole approx. 4 nm wide. The shape of the subunits appears almost mirror-symmetric but the non-homogeneous stain (rather than mass) distribution gives rise to the handedness of the structure (Fig. 2A). The dimensions of the subunits are 8.5 nm close to the outer radius of the enzyme and 7 nm in the radial direction. Side-on views show a thickness of between 5 and 6 nm (Fig. 2B). From the projections in Fig. 2A it cannot be judged whether the five subunits consist of one polypeptide each or of more than one. Gel permeation chromatography yielded an estimate of the molecular mass of 600 kDa for the intact enzyme (Forchhammer etai. 1991b), suggesting that 10 rather than five monomers (molecular mass 50667 Da) make up an assembled molecule. The approximate volume of one morphological unit, assumed to have a uniform thickness of 5 nm, is
Structure of selenocysteine synthase from Escherichia coli and location of tRNA in the seryi-tRNA^^'^-enzyme complex Harald Engelhardt,^* Karl Forchhammer,^ Shirley Muller,^ Kenneth N. Goldie^ and August 'Max-Planck-lnstitut fur Biochemie, D-8033 Martinsried, Germany. ^Lehrstuhl fur f^ilirobiologie der Universitat, D-8000 Munchen 19, Maria-Ward-Strasse la, Germany. ^M.E. Muller-Institute for High-Resolution Electron hAicroscopy at fhe Biocenfre, University of Basel, Ch-4056 Basel, Switzerland. Summary Selenocysteine synthase of Escherichia coii catalyses the biosynthesis of selenocysteine in the form of the aminoacyMRNA complex, the reaction intermediate being aminoacrylyl-tRNA*^" covalently bound to the prosthetic group of the enzyme. Selenocysteine synthase and the specific aminoacrylyl-tRNA^^'' -enzyme complex as well as the isolated seryltRNA^^*^ were investigated in the electron microscope and analysed by means of image processing to a resolution of 2 nm in projection. The stoichiometric composition of the selenocysteine synthase molecule was elucidated by scanning transmission electron microscopic mass determination. The enzyme has a fivefold symmetric structure and consists of 10 monomers arranged in two rings. The tRNA is bound near the margin of the dimeric subunits. Principal component analysis of the tRNA-enzyme complexes revealed that the selenocysteine synthase appears to bind only one seryl-tRNA^*" per dimer, which is consistent with the result of biochemical binding studies.
{Leinfelder et ai, 1988). SeryMRNA^^^ is subsequently converted to selenocysteyl-tRNA^^'' by the novel enzyme selenocysteine synthase through pyridoxal phosphatedependent catalysis (Forchhammer and Bock, 1991). Selenocysteine synthase, the product of gene selA, specifically complexes charged and free tRNA^^'^ and thus functions in tRNA^*"' identification. When the enzyme binds seryl-tRNA^^*^, the substrate is converted to the stable aminoacrylyl-tRNA^^'' intermediate linked to pyridoxal phosphate. Addition of the reactive selenophosphate molecule {Ehrenreich et ai. 1992; Veres et ai, 1992), provided by the protein SELD, generates selenocysteyl-tRNA'^^'' which is released from the enzyme (Forchhammer and Bock, 1991). Selenocysteine synthase is one of the few enzymes that bind aminoacyl-tRNA as a substrate. The tRNA^^"" has a mass and dimensions large enough to be visualized in the electron microscope by appropriate preparation techniques. Usually nucleic acids are rendered visible by heavy-metal shadowing (Kay, 1976), dark-field electron microscopy or labelling by antibodies (Kastner ef ai, 1992). In this study we have successfully applied negative staining to investigate isolated tRNA and tRNA-protein complexes to a resolution of about 2 nm. The stability of the covalent aminoacrylyl-tRNA^^'^-selenocysteine synthase complex enabled us to localize the tRNA^®'' molecules on the enzyme. We also present the projected structure and the stoichiometric composition of the novel enzyme selenocysteine synthase.
Results and Discussion Structure and stoichiometry of the selenocysteine synthase
Introduction The 21st amino acid encoded by the DNA is selenocysleine, a functionally indispensable constituent of several enzymes in eukaryotes, eubacteria and archaebacteria (Bock etai, 1991), In Esc/7enc/i/aco//the biosynthesis of selenocysteine was elucidated recently; it proceeds in two steps. First, L-serine is esterified to selenocysteinespecitic tRNA''^'' by the cellular seryl-tRNA synthetase
Received 1 June, 1992; revised and accepted 22 August, 1992, 'For correspondence. Tel, (89) 85782650; Fax (89) 85782641,
The selenocysteine synthase molecules were imaged in the electron microscope using various heavy-metal salts as negative stains. While ail the stains gave sufficient contrast they had different effects on the orientation of the molecules on the specimen support. Uranyl acetate (pH 4.5) nearly exclusively yielded top-view orientations of the selenocysteine synthase (Fig, 1) while uranyl oxaiate (pH 6.8) resulted in 60-70% side-view orientation. The tRNA-enyzme complexes tended to edge-on orientation throughout with an extreme example for phosphotungstate (pH 6.8) where only 10% of the complexes
3462
H. EngelhardteXa\.
100
200 300 400 500 BOO 700 Mass IkDa)
Fig. 1. Electron microscopic imaging and mass determination ofthe selenocysteine synthase from E. coli. The scale bars represent 100 nm. A. Bright-field electron micrograph of negatively stained selenocysteine synthase showing top-view and side-on view (arrow) projections, B. STEM dark-field micrograph of unstained, freeze-dried moiecules recorded at 350 electrons nm"^. The inset shows the average of 230 molecules, exhibiting a distinct central channel, C. Histogram of mass values extracted from STEM images such as shown in (B), The average of 433±57 kDa is not corrected for beam-induced mass loss which is significant at a recording dose of 300 to 400 electrons/nm^.
appeared in the top-view orientation. In the following uranyt acetate and uranyl oxaiate preparations were analysed. The selenocysteine synthase of E coti is a fivefold symmetric molecule in projection (Figs 1 and 2). Five distinct bi-lobed, cloverleaf-like subunits are connected at thieir bases in a ring-like manner. The enzyme has a diameter of 19 nm and a central hole approx. 4 nm wide. The shape of the subunits appears almost mirror-symmetric but the non-homogeneous stain (rather than mass) distribution gives rise to the handedness of the structure (Fig. 2A). The dimensions of the subunits are 8.5 nm close to the outer radius of the enzyme and 7 nm in the radial direction. Side-on views show a thickness of between 5 and 6 nm (Fig. 2B). From the projections in Fig. 2A it cannot be judged whether the five subunits consist of one polypeptide each or of more than one. Gel permeation chromatography yielded an estimate of the molecular mass of 600 kDa for the intact enzyme (Forchhammer etai. 1991b), suggesting that 10 rather than five monomers (molecular mass 50667 Da) make up an assembled molecule. The approximate volume of one morphological unit, assumed to have a uniform thickness of 5 nm, is
